blunt ended full length α syn  (New England Biolabs)


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    Name:
    DNA Polymerase I Klenow Fragment
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    DNA Polymerase I Klenow Fragment 1 000 units
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    m0210l
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    248
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    1 000 units
    Category:
    DNA Polymerases
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    New England Biolabs blunt ended full length α syn
    DNA Polymerase I Klenow Fragment
    DNA Polymerase I Klenow Fragment 1 000 units
    https://www.bioz.com/result/blunt ended full length α syn/product/New England Biolabs
    Average 92 stars, based on 23205 article reviews
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    blunt ended full length α syn - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease"

    Article Title: An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-180610

    Generation and characterization of inducible α-synuclein (α-Syn) expressing neuronal line. A) Schematic representation of doxycycline-inducible pCW-iFLAG-α-Syn vector used to generate SHSY-5Y stable cell line and immunoblot showing time-dependent induction of FLAG α-synuclein. B) Densitometry analysis of FLAG and endogenous α-synuclein immunocontent normalized to GAPDH. C) Immunofluorescence of oligomer conformations upon α-synuclein expression. Anti-oligomer A11 antibody recognizes all types of oligomers, but not monomers and fibrils in the case of α-synuclein. D) Oligo A11 antibody fluorescence intensity per cells. Measurement from 30 cells from three different fields.
    Figure Legend Snippet: Generation and characterization of inducible α-synuclein (α-Syn) expressing neuronal line. A) Schematic representation of doxycycline-inducible pCW-iFLAG-α-Syn vector used to generate SHSY-5Y stable cell line and immunoblot showing time-dependent induction of FLAG α-synuclein. B) Densitometry analysis of FLAG and endogenous α-synuclein immunocontent normalized to GAPDH. C) Immunofluorescence of oligomer conformations upon α-synuclein expression. Anti-oligomer A11 antibody recognizes all types of oligomers, but not monomers and fibrils in the case of α-synuclein. D) Oligo A11 antibody fluorescence intensity per cells. Measurement from 30 cells from three different fields.

    Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Immunofluorescence, Fluorescence

    2) Product Images from "An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents"

    Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020303

    The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).
    Figure Legend Snippet: The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Techniques Used: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Incubation, BAC Assay

    3) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .
    Figure Legend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Techniques Used: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    4) Product Images from "Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders"

    Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

    Journal: Nature

    doi: 10.1038/nature25449

    Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P
    Figure Legend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Techniques Used: Inhibition, Immunofluorescence, Staining

    5) Product Images from "Effect of Cross-Link Structure on DNA Interstrand Cross-Link Repair Synthesis"

    Article Title: Effect of Cross-Link Structure on DNA Interstrand Cross-Link Repair Synthesis

    Journal:

    doi: 10.1021/tx9000896

    Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template
    Figure Legend Snippet: Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template

    Techniques Used: Primer Extension Assay

    6) Product Images from "Strand displacement synthesis by yeast DNA polymerase ε"

    Article Title: Strand displacement synthesis by yeast DNA polymerase ε

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw556

    Pol ε poorly extends D-loops in comparison to Pol δ but is proficient to extend primed single-stranded DNA using the same substrates under the same conditions. ( A ) In vitro D-loop reactions using a 37-mer oligonucleotide were reconstituted using purified S. cerevisiae proteins as described in Materials and Methods. ( B ) Product analysis of reconstituted D-loop reactions containing either Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) at 0, 2 (not for Klenow), 5 and 10 min extension times. ( C ) Extension of primed single-stranded circular template DNA using a 37-mer oligonucleotide. ( D ) Product analysis of primer extension on denaturing gels of reaction containing Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) each plus or minus 10 nM PCNA/RFC at 0, 2 (not for Klenow), 5 and 10 min extension times. A 100 nt size ladder is shown in the left-most lane.
    Figure Legend Snippet: Pol ε poorly extends D-loops in comparison to Pol δ but is proficient to extend primed single-stranded DNA using the same substrates under the same conditions. ( A ) In vitro D-loop reactions using a 37-mer oligonucleotide were reconstituted using purified S. cerevisiae proteins as described in Materials and Methods. ( B ) Product analysis of reconstituted D-loop reactions containing either Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) at 0, 2 (not for Klenow), 5 and 10 min extension times. ( C ) Extension of primed single-stranded circular template DNA using a 37-mer oligonucleotide. ( D ) Product analysis of primer extension on denaturing gels of reaction containing Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) each plus or minus 10 nM PCNA/RFC at 0, 2 (not for Klenow), 5 and 10 min extension times. A 100 nt size ladder is shown in the left-most lane.

    Techniques Used: In Vitro, Purification

    7) Product Images from "Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes"

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks316

    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.
    Figure Legend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Techniques Used: Purification, Polymerase Chain Reaction, Construct

    8) Product Images from "An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum"

    Article Title: An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum

    Journal: bioRxiv

    doi: 10.1101/816504

    Proof-of-concept to establish successful transfer of large DNA fragments containing interspersed regions of AT-rich regulatory elements to a linear vector framework. (A) . The schematic shows 7.5 kb and 9.5 kb fragments to be released from extant circular vectors pSN372 and pMG1847, respectively, for assembly into linear vectors. These fragments contain TetR- or TetR-DOZI-based translation regulation modules and a transcriptional unit in which expression of a FLuc reporter CDS is translationally controlled by TetR aptamers located in either the 5’- UTR only or both 5’- and 3’- UTR s. (B) Strategy used to transfer the respective pSN372- and pMG1847-derived fragments into linear plasmids. The original pJAZZ-OC vector (Lucigen) was modified with a multi-cloning site gene block to create pSwing. To facilitate Gibson assembly, pSwing can be digested with restriction enzymes to expose regions homologous to cut pSN372- and pMG1847-derived fragments (red and green). (C) . Restriction digestion analysis confirming proper topological assembly of pSwing, pSN372L and pSN1847L. For pSN1847L, several plasmids that do not contain the expected insert, and likely corresponding to pSwing, are indicated in red font.
    Figure Legend Snippet: Proof-of-concept to establish successful transfer of large DNA fragments containing interspersed regions of AT-rich regulatory elements to a linear vector framework. (A) . The schematic shows 7.5 kb and 9.5 kb fragments to be released from extant circular vectors pSN372 and pMG1847, respectively, for assembly into linear vectors. These fragments contain TetR- or TetR-DOZI-based translation regulation modules and a transcriptional unit in which expression of a FLuc reporter CDS is translationally controlled by TetR aptamers located in either the 5’- UTR only or both 5’- and 3’- UTR s. (B) Strategy used to transfer the respective pSN372- and pMG1847-derived fragments into linear plasmids. The original pJAZZ-OC vector (Lucigen) was modified with a multi-cloning site gene block to create pSwing. To facilitate Gibson assembly, pSwing can be digested with restriction enzymes to expose regions homologous to cut pSN372- and pMG1847-derived fragments (red and green). (C) . Restriction digestion analysis confirming proper topological assembly of pSwing, pSN372L and pSN1847L. For pSN1847L, several plasmids that do not contain the expected insert, and likely corresponding to pSwing, are indicated in red font.

    Techniques Used: Plasmid Preparation, Expressing, Derivative Assay, Modification, Clone Assay, Blocking Assay

    9) Product Images from "Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize"

    Article Title: Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000582

    Circular CRM1 RNAs induce chromatin loops in the centromere. (A) Anti-S9.6 RIP-qPCR was used to confirm the R-loop formation by 354-, 607-, and 277- to 296-nt circular CRM1 RNAs. Zm00001d007960 RNA was used as a negative control, and rRNA was used as a positive control. Chromatin-binding RNA was used for RIP. Actin was used as an internal reference gene. (B) Regions chosen for detecting the ssDNA sites are marked as 85–1, 253–1, 269–1, and 269–2. (C) ssDNA sites in CRM1 were checked using an S1 nuclease treatment of the nuclear DNA. DNA with no S1 nuclease treatment was used as a control template. The 607-left sequence was used as an internal reference gene. (D and E) Potential chromatin loops were induced by circular RNA inside a single CRM1 element (D) and between two CRM1 elements (E). Red, green, and yellow lines represent the 85-, 269-, and 253-bp regions, respectively. Black lines represent sequences on the left side of the 85-bp sequence and the right side of the 269-bp sequence. The blue ovals represent circular CRM1 RNAs. ①, ‘①’, ②, and ③ represent the broken ends on the two sides of the 253-bp sequence, the left side of the 85-bp sequence, and the right side of the 269-bp sequence. (F) 3C-PCR confirms the potential ligations of chromatin loops after DpnII digestion. The left panel shows the PCR results in the undigested, unligated samples and 3C samples under potential ligation forms. The right panel shows the sequences from the bands on the left, including the expected sequences, the first and the second part of the expected sequences, and the amplified sequences. (G and H) 3C-qPCR shows chromatin interactions inside a single CRM1 element (G) and between two CRM1 elements (H). The interaction frequencies between two DpnII -digested fragments were normalized to the 3C control template from the digested and ligated centromeric BAC clone and an internal reference gene, SAM . In (A), (C), (G), and (H), the columns and error bars represent the relative value and standard error of the means ( n = 3). In (A) and (C), the P values were determined using a Student t test: * P
    Figure Legend Snippet: Circular CRM1 RNAs induce chromatin loops in the centromere. (A) Anti-S9.6 RIP-qPCR was used to confirm the R-loop formation by 354-, 607-, and 277- to 296-nt circular CRM1 RNAs. Zm00001d007960 RNA was used as a negative control, and rRNA was used as a positive control. Chromatin-binding RNA was used for RIP. Actin was used as an internal reference gene. (B) Regions chosen for detecting the ssDNA sites are marked as 85–1, 253–1, 269–1, and 269–2. (C) ssDNA sites in CRM1 were checked using an S1 nuclease treatment of the nuclear DNA. DNA with no S1 nuclease treatment was used as a control template. The 607-left sequence was used as an internal reference gene. (D and E) Potential chromatin loops were induced by circular RNA inside a single CRM1 element (D) and between two CRM1 elements (E). Red, green, and yellow lines represent the 85-, 269-, and 253-bp regions, respectively. Black lines represent sequences on the left side of the 85-bp sequence and the right side of the 269-bp sequence. The blue ovals represent circular CRM1 RNAs. ①, ‘①’, ②, and ③ represent the broken ends on the two sides of the 253-bp sequence, the left side of the 85-bp sequence, and the right side of the 269-bp sequence. (F) 3C-PCR confirms the potential ligations of chromatin loops after DpnII digestion. The left panel shows the PCR results in the undigested, unligated samples and 3C samples under potential ligation forms. The right panel shows the sequences from the bands on the left, including the expected sequences, the first and the second part of the expected sequences, and the amplified sequences. (G and H) 3C-qPCR shows chromatin interactions inside a single CRM1 element (G) and between two CRM1 elements (H). The interaction frequencies between two DpnII -digested fragments were normalized to the 3C control template from the digested and ligated centromeric BAC clone and an internal reference gene, SAM . In (A), (C), (G), and (H), the columns and error bars represent the relative value and standard error of the means ( n = 3). In (A) and (C), the P values were determined using a Student t test: * P

    Techniques Used: Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Binding Assay, Sequencing, Polymerase Chain Reaction, Ligation, Amplification, BAC Assay

    10) Product Images from "Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes"

    Article Title: Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes

    Journal: bioRxiv

    doi: 10.1101/2020.01.27.921452

    The VEX complex associates with both the active-VSG and the Spliced Leader ( SL )-locus in a cell cycle and developmental stage-dependent manner. a-b, Immunofluorescence-based colocalisation studies of VEX1 myc / Pol I and GFP VEX2 / tSNAP myc in bloodstream form cells. tSNAP and Pol I were used as markers for the SL-RNA and VSG transcription compartments, respectively. The stacked bar graphs depict proportions of nuclei with overlapping, adjacent or separate signals and values are averages of two independent experiments (≥100 nuclei for G1 and S phase cells); detailed n and p values are provided in Data S1 sheet 3. c, VEX1 myc chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) analysis. The circle plot represents log2 fold change of ChIP versus Input of non-overlapping 1 kbp bins of the 11 megabase chromosomes; outside track shows tandem arrays (red) and the SL-RNA locus (black). An inset zooming on the SL-RNA locus is depicted: metagene plot (left-hand side) and heat-map (right-hand side) of SL-gene loci. Bin size 300 bp. d, Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar marker (NOG1) in bloodstream forms. e, Localisation of tSNAP GFP and colocalisation studies of VEX1 myc or myc VEX2 and Pol I in procyclic forms (insect-stage), using immunofluorescence. Procyclic forms do not express VSGs whereas procyclins are the major surface glycoprotein. Images in a-b / d-e were obtained with super resolution microscopy and correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. f, Western-blot analysis of VEX1 myc , myc VEX2 and tSNAP myc before and after sinefungin treatment (5 μg ml -1 for 30 min at 37°C), which blocks trans -splicing in trypanosomes. Data in a-b and d-f are representative of at least two independent biological replicates and two independent experiments.
    Figure Legend Snippet: The VEX complex associates with both the active-VSG and the Spliced Leader ( SL )-locus in a cell cycle and developmental stage-dependent manner. a-b, Immunofluorescence-based colocalisation studies of VEX1 myc / Pol I and GFP VEX2 / tSNAP myc in bloodstream form cells. tSNAP and Pol I were used as markers for the SL-RNA and VSG transcription compartments, respectively. The stacked bar graphs depict proportions of nuclei with overlapping, adjacent or separate signals and values are averages of two independent experiments (≥100 nuclei for G1 and S phase cells); detailed n and p values are provided in Data S1 sheet 3. c, VEX1 myc chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) analysis. The circle plot represents log2 fold change of ChIP versus Input of non-overlapping 1 kbp bins of the 11 megabase chromosomes; outside track shows tandem arrays (red) and the SL-RNA locus (black). An inset zooming on the SL-RNA locus is depicted: metagene plot (left-hand side) and heat-map (right-hand side) of SL-gene loci. Bin size 300 bp. d, Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar marker (NOG1) in bloodstream forms. e, Localisation of tSNAP GFP and colocalisation studies of VEX1 myc or myc VEX2 and Pol I in procyclic forms (insect-stage), using immunofluorescence. Procyclic forms do not express VSGs whereas procyclins are the major surface glycoprotein. Images in a-b / d-e were obtained with super resolution microscopy and correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. f, Western-blot analysis of VEX1 myc , myc VEX2 and tSNAP myc before and after sinefungin treatment (5 μg ml -1 for 30 min at 37°C), which blocks trans -splicing in trypanosomes. Data in a-b and d-f are representative of at least two independent biological replicates and two independent experiments.

    Techniques Used: Immunofluorescence, Chromatin Immunoprecipitation, Next-Generation Sequencing, Marker, Microscopy, Staining, Western Blot

    The active VSG expression site (ES) stably interacts with the spliced leader RNA (SL) array. a, Hi-C (virtual 4C) analysis, viewpoints: active VSG gene in ES1 ( VSG-2 , top panel) and silent VSG gene in ES 3 ( VSG-6 , bottom panel). Relative interaction frequencies between the viewpoint and the 11 megabase chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)– 3’(haplotype B). Bin size 50 kb. b, Virtual 4C analysis, viewpoints: active VSG gene in ES 1 and inactive VSG genes in ES 3, 4, 5, 7, 11, 13 and 15. Relative interaction frequencies between the viewpoint and the SL-RNA locus on the right arm of chr. 9 is plotted. Bin size 20 kb. The analyses in a - b are based on Hi-C experiments with VSG-2 expressing cells (n=2, average interaction frequencies are shown). c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA locus marker – SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. The stacked bar graph depicts proportions of G1 or S phase nuclei with overlapping, adjacent or separate signals for the SL-RNA and VSG transcription compartments. Values are averages of three independent experiments and representative of two independent biological replicates (≥100 G1 or S phase nuclei); error bars, SD. Detailed n and p values are provided in Data S1 sheet 3. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices; scale bars 2 μm. N, nucleus; K, kinetoplast (mitochondrial genome).
    Figure Legend Snippet: The active VSG expression site (ES) stably interacts with the spliced leader RNA (SL) array. a, Hi-C (virtual 4C) analysis, viewpoints: active VSG gene in ES1 ( VSG-2 , top panel) and silent VSG gene in ES 3 ( VSG-6 , bottom panel). Relative interaction frequencies between the viewpoint and the 11 megabase chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)– 3’(haplotype B). Bin size 50 kb. b, Virtual 4C analysis, viewpoints: active VSG gene in ES 1 and inactive VSG genes in ES 3, 4, 5, 7, 11, 13 and 15. Relative interaction frequencies between the viewpoint and the SL-RNA locus on the right arm of chr. 9 is plotted. Bin size 20 kb. The analyses in a - b are based on Hi-C experiments with VSG-2 expressing cells (n=2, average interaction frequencies are shown). c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA locus marker – SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. The stacked bar graph depicts proportions of G1 or S phase nuclei with overlapping, adjacent or separate signals for the SL-RNA and VSG transcription compartments. Values are averages of three independent experiments and representative of two independent biological replicates (≥100 G1 or S phase nuclei); error bars, SD. Detailed n and p values are provided in Data S1 sheet 3. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices; scale bars 2 μm. N, nucleus; K, kinetoplast (mitochondrial genome).

    Techniques Used: Expressing, Stable Transfection, Hi-C, Immunofluorescence, Marker, Microscopy, Staining

    Pol I and tSNAP expression and localisation following knockdown of the VEX complex. a, Immunofluorescence-based analysis of VSG expression following tetracycline (Tet) inducible VEX1 knockdown, VEX2 knockdown or VEX1/VEX2 knockdown. VSG-2 (magenta) is the active- VSG and VSG-6 (green) is a silent- VSG in this strain. The stacked bar graph depicts percentages of VSG-2 single positive cells and VSG-2/VSG-6 double positive cells; values are averages of two independent experiments and two biological replicates. DNA was counter-stained with DAPI; scale bar 2 μm. b, Western-blot analysis of VEX2, Pol I, tSNAP myc , VSG-6 and VSG-2 expression following VEX1, VEX2 or VEX1/VEX2 knockdown. EF1α was used as a loading control. The data is representative of two independent experiments and two biological replicates. c-d , Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar and active- VSG marker (Pol I, large subunit). The stacked bar graph in c depicts proportions of G1 nuclei with tSNAP myc / Pol I overlapping, adjacent or separate signals following tetracycline (Tet) inducible VEX1 (48 h), VEX2 (12 h) or VEX1/VEX2 knockdown (12 h). tSNAP myc / active- VSG localisation were not monitored beyond 12 h following VEX2 and VEX1/2 knockdown as Pol I signal drops below detection at later time-points. The values are averages of two independent experiments and two biological replicates (≥100 G1 nuclei). In the box plot in d , the distance between the edges of the ESB and tSNAP foci was measured in > 81 G1 nuclei. The centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. In a/c , error bars, SD. In c-d , knockdown conditions were compared to parental cells using two-tailed paired ( c ) or unpaired ( d ) Student’s t -tests: *, p
    Figure Legend Snippet: Pol I and tSNAP expression and localisation following knockdown of the VEX complex. a, Immunofluorescence-based analysis of VSG expression following tetracycline (Tet) inducible VEX1 knockdown, VEX2 knockdown or VEX1/VEX2 knockdown. VSG-2 (magenta) is the active- VSG and VSG-6 (green) is a silent- VSG in this strain. The stacked bar graph depicts percentages of VSG-2 single positive cells and VSG-2/VSG-6 double positive cells; values are averages of two independent experiments and two biological replicates. DNA was counter-stained with DAPI; scale bar 2 μm. b, Western-blot analysis of VEX2, Pol I, tSNAP myc , VSG-6 and VSG-2 expression following VEX1, VEX2 or VEX1/VEX2 knockdown. EF1α was used as a loading control. The data is representative of two independent experiments and two biological replicates. c-d , Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar and active- VSG marker (Pol I, large subunit). The stacked bar graph in c depicts proportions of G1 nuclei with tSNAP myc / Pol I overlapping, adjacent or separate signals following tetracycline (Tet) inducible VEX1 (48 h), VEX2 (12 h) or VEX1/VEX2 knockdown (12 h). tSNAP myc / active- VSG localisation were not monitored beyond 12 h following VEX2 and VEX1/2 knockdown as Pol I signal drops below detection at later time-points. The values are averages of two independent experiments and two biological replicates (≥100 G1 nuclei). In the box plot in d , the distance between the edges of the ESB and tSNAP foci was measured in > 81 G1 nuclei. The centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. In a/c , error bars, SD. In c-d , knockdown conditions were compared to parental cells using two-tailed paired ( c ) or unpaired ( d ) Student’s t -tests: *, p

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot, Marker, Two Tailed Test

    The exclusive association between the active VSG and the SL -locus is VEX2-dependent. a-b, Immunofluorescence and super resolution microscopy based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and Pol I (nucleolus and active-VSG transcription compartment) following VEX1, VEX2 and VEX1/VEX2 knockdown. a, Representative images, all nuclei are G1. On the right-hand side, two representative histograms depict the distribution of signal intensity across the distance indicated by the dashed lines. Images correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. b, The box plot depicts the distance between the active-VSG and the nearest SL-RNA transcription compartment (≥ 81 G1 nuclei) following VEX1, VEX2 and VEX1/VEX2 knockdown; the centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. The distances for each knockdown condition were compared to the parental cell line using a two-tailed unpaired Student’s t -test. c, Hi-C (virtual 4C) analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the VSG expression sites are shown before and after VEX2 knockdown. Each dot represents the average value for one expression site. Bin size 20 kb. d, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and VSG expression sites is shown. Bin size 20 kb. e, Virtual 4C analysis, viewpoint: EP1 (chr. 10) or GPEET gene array (chr. 6). Relative interaction frequencies between the viewpoint and the SL-RNA locus are plotted. Bin size 20 kb. The analyses in c to e are based on Hi-C experiments with cells before and 24 h after VEX2 knockdown (n=3, the average is shown). f, Schematic model for monogenic VSG expression. A strong inter-chromosomal interaction between the SL -array and the active VSG gene facilitates spatial integration of transcription and mRNA maturation. VEX1 and VEX2 are primarily SL - and active-VSG associated, respectively, and sustain monogenic VSG expression by excluding other VSGs. The VSG-SL organelle is reconfigured upon activation of a different VSG.
    Figure Legend Snippet: The exclusive association between the active VSG and the SL -locus is VEX2-dependent. a-b, Immunofluorescence and super resolution microscopy based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and Pol I (nucleolus and active-VSG transcription compartment) following VEX1, VEX2 and VEX1/VEX2 knockdown. a, Representative images, all nuclei are G1. On the right-hand side, two representative histograms depict the distribution of signal intensity across the distance indicated by the dashed lines. Images correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. b, The box plot depicts the distance between the active-VSG and the nearest SL-RNA transcription compartment (≥ 81 G1 nuclei) following VEX1, VEX2 and VEX1/VEX2 knockdown; the centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. The distances for each knockdown condition were compared to the parental cell line using a two-tailed unpaired Student’s t -test. c, Hi-C (virtual 4C) analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the VSG expression sites are shown before and after VEX2 knockdown. Each dot represents the average value for one expression site. Bin size 20 kb. d, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and VSG expression sites is shown. Bin size 20 kb. e, Virtual 4C analysis, viewpoint: EP1 (chr. 10) or GPEET gene array (chr. 6). Relative interaction frequencies between the viewpoint and the SL-RNA locus are plotted. Bin size 20 kb. The analyses in c to e are based on Hi-C experiments with cells before and 24 h after VEX2 knockdown (n=3, the average is shown). f, Schematic model for monogenic VSG expression. A strong inter-chromosomal interaction between the SL -array and the active VSG gene facilitates spatial integration of transcription and mRNA maturation. VEX1 and VEX2 are primarily SL - and active-VSG associated, respectively, and sustain monogenic VSG expression by excluding other VSGs. The VSG-SL organelle is reconfigured upon activation of a different VSG.

    Techniques Used: Immunofluorescence, Microscopy, Staining, Two Tailed Test, Hi-C, Expressing, Activation Assay

    Genome-wide interaction frequencies of VSG genes in expression sites and the SL-RNA locus. a, Hi-C (virtual 4C) analysis with locations of viewpoints marked by pink boxes. Viewpoints VSG ES 4, 7, 11 and 17 are located on intermediate chromosomes that are not depicted in this figure. Interaction frequencies between each viewpoint and the 11 mega-base chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions of each chromosome are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)–3’(haplotype B). Bin size 50 kb. Virtual 4C analyses in a-b are based on Hi-C experiments of VSG-2 expressing cells (n=2, the average is shown). b, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the active VSG ES 1 (cyan) and inactive VSG ESs (magenta) is shown. Each dot represents the average value for one expression site. Bin size 20 kb. c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices and are representative of two biological replicates and three independent experiments; scale bars 2 μm.
    Figure Legend Snippet: Genome-wide interaction frequencies of VSG genes in expression sites and the SL-RNA locus. a, Hi-C (virtual 4C) analysis with locations of viewpoints marked by pink boxes. Viewpoints VSG ES 4, 7, 11 and 17 are located on intermediate chromosomes that are not depicted in this figure. Interaction frequencies between each viewpoint and the 11 mega-base chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions of each chromosome are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)–3’(haplotype B). Bin size 50 kb. Virtual 4C analyses in a-b are based on Hi-C experiments of VSG-2 expressing cells (n=2, the average is shown). b, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the active VSG ES 1 (cyan) and inactive VSG ESs (magenta) is shown. Each dot represents the average value for one expression site. Bin size 20 kb. c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices and are representative of two biological replicates and three independent experiments; scale bars 2 μm.

    Techniques Used: Genome Wide, Expressing, Hi-C, Immunofluorescence, Marker, Microscopy, Staining

    11) Product Images from "Construction of a circular single-stranded DNA template containing a defined lesion"

    Article Title: Construction of a circular single-stranded DNA template containing a defined lesion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2009.03.006

    ( A ) Sequence of pSOcpd surrounding the cis-syn CPD (indicated as T-T in bold font). The binding site of the labeled primer M13-TT, is shown as an arrow. ( B ) In vitro DNA replication assay with pSOcpd. The TLS reactions were performed in the presence of pol I (Kf) (lane 1), T7 DNA polymerase (lane 2), pol V (R391) + RecA protein in the absence (lane 3), or presence of the β-clamp and γ-clamp loader complex (lane 4), or human polη (lane 5). The position of the labeled primer (lane 6), is shown on the right of the gel (P), while the local template sequence context and position of the T-T CPD (in bold font) is shown on the left side of the gel.
    Figure Legend Snippet: ( A ) Sequence of pSOcpd surrounding the cis-syn CPD (indicated as T-T in bold font). The binding site of the labeled primer M13-TT, is shown as an arrow. ( B ) In vitro DNA replication assay with pSOcpd. The TLS reactions were performed in the presence of pol I (Kf) (lane 1), T7 DNA polymerase (lane 2), pol V (R391) + RecA protein in the absence (lane 3), or presence of the β-clamp and γ-clamp loader complex (lane 4), or human polη (lane 5). The position of the labeled primer (lane 6), is shown on the right of the gel (P), while the local template sequence context and position of the T-T CPD (in bold font) is shown on the left side of the gel.

    Techniques Used: Sequencing, Binding Assay, Labeling, In Vitro

    12) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    13) Product Images from "Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease"

    Article Title: Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-170342

    Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.
    Figure Legend Snippet: Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.

    Techniques Used: Binding Assay, Stable Transfection, Plasmid Preparation, Immunofluorescence, Proximity Ligation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, In Vitro, Co-Elution Assay, Recombinant, Labeling

    14) Product Images from "Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells †"

    Article Title: Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells †

    Journal: Biochemistry

    doi: 10.1021/bi902169q

    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.
    Figure Legend Snippet: A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.

    Techniques Used: Plasmid Preparation, Ligation, Blocking Assay, Labeling

    15) Product Images from "Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease"

    Article Title: Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-170342

    α-Syn DNA nicking activity may be mediated by its oxidation. A) Exposure of recombinant α-Syn to 1 O 2 , generated by exposing flavonoid Bengal red to UVB radiation, enhanced its DNA nicking activity with plasmid scDNA in vitro . The products were analyzed by agarose gel electrophoresis. The histogram represents quantification of DNA fragmentation. B) SHSY-5Y cells were exposed to similarly oxidized α-Syn, and DNA damage was quantified by alkaline Comet assay.
    Figure Legend Snippet: α-Syn DNA nicking activity may be mediated by its oxidation. A) Exposure of recombinant α-Syn to 1 O 2 , generated by exposing flavonoid Bengal red to UVB radiation, enhanced its DNA nicking activity with plasmid scDNA in vitro . The products were analyzed by agarose gel electrophoresis. The histogram represents quantification of DNA fragmentation. B) SHSY-5Y cells were exposed to similarly oxidized α-Syn, and DNA damage was quantified by alkaline Comet assay.

    Techniques Used: Activity Assay, Recombinant, Generated, Plasmid Preparation, In Vitro, Agarose Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis

    Increased α-Syn expression causes DNA breaks in neurons synergistically with pro-oxidant metals. A, B) Alkaline Comet assay in iFLAG-α-Syn SHSY-5Y cells exposed to 200 μM FeSO 4 or CuSO 4 . α-Syn was induced with Dox for 48 h. Metal salts alone at the same concentration caused only moderate increases in strand breaks. C, D) Semi-quantitative LA-PCR assay for genomic DNA isolated from pCW-iFLAG-α-Syn SHSY-5Y cells in the presence of FeSO 4 or CuSO 4 . *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Increased α-Syn expression causes DNA breaks in neurons synergistically with pro-oxidant metals. A, B) Alkaline Comet assay in iFLAG-α-Syn SHSY-5Y cells exposed to 200 μM FeSO 4 or CuSO 4 . α-Syn was induced with Dox for 48 h. Metal salts alone at the same concentration caused only moderate increases in strand breaks. C, D) Semi-quantitative LA-PCR assay for genomic DNA isolated from pCW-iFLAG-α-Syn SHSY-5Y cells in the presence of FeSO 4 or CuSO 4 . *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Expressing, Alkaline Single Cell Gel Electrophoresis, Concentration Assay, Polymerase Chain Reaction, Isolation

    MD simulation: α-Syn N-terminal residues may be involved in DNA binding. Protein-DNA docking Model 1 demonstrating binding of α-Syn (PDB: 1XQ8) N-terminal amino acid residues Glu-35, Ser-42, Thr-54 to the crystal structure of d(CCGGTACCGG) as a B-DNA duplex (PDB: 3IXN). This structure model represents the best structure from the biggest cluster after the refinement process. Structures were analyzed using PyMOL Molecular Graphics System, Version 1.7.4.5 Schrödinger, LLC.
    Figure Legend Snippet: MD simulation: α-Syn N-terminal residues may be involved in DNA binding. Protein-DNA docking Model 1 demonstrating binding of α-Syn (PDB: 1XQ8) N-terminal amino acid residues Glu-35, Ser-42, Thr-54 to the crystal structure of d(CCGGTACCGG) as a B-DNA duplex (PDB: 3IXN). This structure model represents the best structure from the biggest cluster after the refinement process. Structures were analyzed using PyMOL Molecular Graphics System, Version 1.7.4.5 Schrödinger, LLC.

    Techniques Used: Binding Assay

    Model illustrating how α-Syn-induced DNA breaks contributes to neuronal apoptosis in PD. The role of pro-oxidant Fe or ROS in promoting α-Syn misfolding and oxidation, which could exacerbate its DNA nicking activity.
    Figure Legend Snippet: Model illustrating how α-Syn-induced DNA breaks contributes to neuronal apoptosis in PD. The role of pro-oxidant Fe or ROS in promoting α-Syn misfolding and oxidation, which could exacerbate its DNA nicking activity.

    Techniques Used: Activity Assay

    Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.
    Figure Legend Snippet: Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.

    Techniques Used: Binding Assay, Stable Transfection, Plasmid Preparation, Immunofluorescence, Proximity Ligation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, In Vitro, Co-Elution Assay, Recombinant, Labeling

    Recombinant α-Syn nicks naked DNA in vitro , which is enhanced by its misfolding/oligomerization. A) Agarose gel electrophoresis showing plasmid scDNA cleavage by recombinant α-Syn. B) Assessment of SSBs and DSBs in scDNA induced by α-Syn. Values are expressed as SSBs and DSBs induced per μg of scDNA. C) Recombinant α-Syn was incubated with constant stirring to cause its aggregation, which was monitored by Thio T fluorescence analysis with an aliquot of α-Syn taken at various time intervals. D) The α-Syn aliquots were also analyzed for their DNA nicking activity with plasmid DNA by agarose gel electrophoresis. E) DNA breaks were quantified. F) Misfolding/β-sheet formation in α-Syn upon stirring as confirmed by CD spectroscopy.
    Figure Legend Snippet: Recombinant α-Syn nicks naked DNA in vitro , which is enhanced by its misfolding/oligomerization. A) Agarose gel electrophoresis showing plasmid scDNA cleavage by recombinant α-Syn. B) Assessment of SSBs and DSBs in scDNA induced by α-Syn. Values are expressed as SSBs and DSBs induced per μg of scDNA. C) Recombinant α-Syn was incubated with constant stirring to cause its aggregation, which was monitored by Thio T fluorescence analysis with an aliquot of α-Syn taken at various time intervals. D) The α-Syn aliquots were also analyzed for their DNA nicking activity with plasmid DNA by agarose gel electrophoresis. E) DNA breaks were quantified. F) Misfolding/β-sheet formation in α-Syn upon stirring as confirmed by CD spectroscopy.

    Techniques Used: Recombinant, In Vitro, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Fluorescence, Activity Assay, Spectroscopy

    DNA damage in neurons generated from normal and PD patient-derived SNCA -tri iPSC cells. A) Phase contrast image demonstrating generation of NPCs from iPSCs: (a) SNCA- tri iPSCs cultured in MEF feeder layer, (b) SNCA- tri iPSCs cultured in feeder free layer, (c) day 2, (d) day 4, (e) day 6 of neural induction for NPC derivation, (f) NPC at passage 3. The iPSC specific marker Oct4 and neural precursor markers nestin analyzed by immunoblotting (B). C) Immunofluorescence characterization with nestin and α-Syn protein expression. D) α-SYN mRNA quantitation in control versus SNCA-tri iPSC and NPC cells. E, F) LA-PCR analysis of genomic DNA isolated from control or SNCA -tri NPC cells exposed to 200 μM FeSO 4 or CuSO 4 . *** p ≤ 0.001.
    Figure Legend Snippet: DNA damage in neurons generated from normal and PD patient-derived SNCA -tri iPSC cells. A) Phase contrast image demonstrating generation of NPCs from iPSCs: (a) SNCA- tri iPSCs cultured in MEF feeder layer, (b) SNCA- tri iPSCs cultured in feeder free layer, (c) day 2, (d) day 4, (e) day 6 of neural induction for NPC derivation, (f) NPC at passage 3. The iPSC specific marker Oct4 and neural precursor markers nestin analyzed by immunoblotting (B). C) Immunofluorescence characterization with nestin and α-Syn protein expression. D) α-SYN mRNA quantitation in control versus SNCA-tri iPSC and NPC cells. E, F) LA-PCR analysis of genomic DNA isolated from control or SNCA -tri NPC cells exposed to 200 μM FeSO 4 or CuSO 4 . *** p ≤ 0.001.

    Techniques Used: Generated, Derivative Assay, Cell Culture, Marker, Immunofluorescence, Expressing, Quantitation Assay, Polymerase Chain Reaction, Isolation

    16) Product Images from "An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease 1"

    Article Title: An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease 1

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-180610

    Generation and characterization of inducible α-synuclein (α-Syn) expressing neuronal line. A) Schematic representation of doxycycline-inducible pCW-iFLAG-α-Syn vector used to generate SHSY-5Y stable cell line and immunoblot showing time-dependent induction of FLAG α-synuclein. B) Densitometry analysis of FLAG and endogenous α-synuclein immunocontent normalized to GAPDH. C) Immunofluorescence of oligomer conformations upon α-synuclein expression. Anti-oligomer A11 antibody recognizes all types of oligomers, but not monomers and fibrils in the case of α-synuclein. D) Oligo A11 antibody fluorescence intensity per cells. Measurement from 30 cells from three different fields.
    Figure Legend Snippet: Generation and characterization of inducible α-synuclein (α-Syn) expressing neuronal line. A) Schematic representation of doxycycline-inducible pCW-iFLAG-α-Syn vector used to generate SHSY-5Y stable cell line and immunoblot showing time-dependent induction of FLAG α-synuclein. B) Densitometry analysis of FLAG and endogenous α-synuclein immunocontent normalized to GAPDH. C) Immunofluorescence of oligomer conformations upon α-synuclein expression. Anti-oligomer A11 antibody recognizes all types of oligomers, but not monomers and fibrils in the case of α-synuclein. D) Oligo A11 antibody fluorescence intensity per cells. Measurement from 30 cells from three different fields.

    Techniques Used: Expressing, Plasmid Preparation, Stable Transfection, Immunofluorescence, Fluorescence

    17) Product Images from "Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes"

    Article Title: Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes

    Journal: bioRxiv

    doi: 10.1101/2020.01.27.921452

    The VEX complex associates with both the active-VSG and the Spliced Leader ( SL )-locus in a cell cycle and developmental stage-dependent manner. a-b, Immunofluorescence-based colocalisation studies of VEX1 myc / Pol I and GFP VEX2 / tSNAP myc in bloodstream form cells. tSNAP and Pol I were used as markers for the SL-RNA and VSG transcription compartments, respectively. The stacked bar graphs depict proportions of nuclei with overlapping, adjacent or separate signals and values are averages of two independent experiments (≥100 nuclei for G1 and S phase cells); detailed n and p values are provided in Data S1 sheet 3. c, VEX1 myc chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) analysis. The circle plot represents log2 fold change of ChIP versus Input of non-overlapping 1 kbp bins of the 11 megabase chromosomes; outside track shows tandem arrays (red) and the SL-RNA locus (black). An inset zooming on the SL-RNA locus is depicted: metagene plot (left-hand side) and heat-map (right-hand side) of SL-gene loci. Bin size 300 bp. d, Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar marker (NOG1) in bloodstream forms. e, Localisation of tSNAP GFP and colocalisation studies of VEX1 myc or myc VEX2 and Pol I in procyclic forms (insect-stage), using immunofluorescence. Procyclic forms do not express VSGs whereas procyclins are the major surface glycoprotein. Images in a-b / d-e were obtained with super resolution microscopy and correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. f, Western-blot analysis of VEX1 myc , myc VEX2 and tSNAP myc before and after sinefungin treatment (5 μg ml -1 for 30 min at 37°C), which blocks trans -splicing in trypanosomes. Data in a-b and d-f are representative of at least two independent biological replicates and two independent experiments.
    Figure Legend Snippet: The VEX complex associates with both the active-VSG and the Spliced Leader ( SL )-locus in a cell cycle and developmental stage-dependent manner. a-b, Immunofluorescence-based colocalisation studies of VEX1 myc / Pol I and GFP VEX2 / tSNAP myc in bloodstream form cells. tSNAP and Pol I were used as markers for the SL-RNA and VSG transcription compartments, respectively. The stacked bar graphs depict proportions of nuclei with overlapping, adjacent or separate signals and values are averages of two independent experiments (≥100 nuclei for G1 and S phase cells); detailed n and p values are provided in Data S1 sheet 3. c, VEX1 myc chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) analysis. The circle plot represents log2 fold change of ChIP versus Input of non-overlapping 1 kbp bins of the 11 megabase chromosomes; outside track shows tandem arrays (red) and the SL-RNA locus (black). An inset zooming on the SL-RNA locus is depicted: metagene plot (left-hand side) and heat-map (right-hand side) of SL-gene loci. Bin size 300 bp. d, Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar marker (NOG1) in bloodstream forms. e, Localisation of tSNAP GFP and colocalisation studies of VEX1 myc or myc VEX2 and Pol I in procyclic forms (insect-stage), using immunofluorescence. Procyclic forms do not express VSGs whereas procyclins are the major surface glycoprotein. Images in a-b / d-e were obtained with super resolution microscopy and correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. f, Western-blot analysis of VEX1 myc , myc VEX2 and tSNAP myc before and after sinefungin treatment (5 μg ml -1 for 30 min at 37°C), which blocks trans -splicing in trypanosomes. Data in a-b and d-f are representative of at least two independent biological replicates and two independent experiments.

    Techniques Used: Immunofluorescence, Chromatin Immunoprecipitation, Next-Generation Sequencing, Marker, Microscopy, Staining, Western Blot

    The active VSG expression site (ES) stably interacts with the spliced leader RNA (SL) array. a, Hi-C (virtual 4C) analysis, viewpoints: active VSG gene in ES1 ( VSG-2 , top panel) and silent VSG gene in ES 3 ( VSG-6 , bottom panel). Relative interaction frequencies between the viewpoint and the 11 megabase chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)– 3’(haplotype B). Bin size 50 kb. b, Virtual 4C analysis, viewpoints: active VSG gene in ES 1 and inactive VSG genes in ES 3, 4, 5, 7, 11, 13 and 15. Relative interaction frequencies between the viewpoint and the SL-RNA locus on the right arm of chr. 9 is plotted. Bin size 20 kb. The analyses in a - b are based on Hi-C experiments with VSG-2 expressing cells (n=2, average interaction frequencies are shown). c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA locus marker – SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. The stacked bar graph depicts proportions of G1 or S phase nuclei with overlapping, adjacent or separate signals for the SL-RNA and VSG transcription compartments. Values are averages of three independent experiments and representative of two independent biological replicates (≥100 G1 or S phase nuclei); error bars, SD. Detailed n and p values are provided in Data S1 sheet 3. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices; scale bars 2 μm. N, nucleus; K, kinetoplast (mitochondrial genome).
    Figure Legend Snippet: The active VSG expression site (ES) stably interacts with the spliced leader RNA (SL) array. a, Hi-C (virtual 4C) analysis, viewpoints: active VSG gene in ES1 ( VSG-2 , top panel) and silent VSG gene in ES 3 ( VSG-6 , bottom panel). Relative interaction frequencies between the viewpoint and the 11 megabase chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)– 3’(haplotype B). Bin size 50 kb. b, Virtual 4C analysis, viewpoints: active VSG gene in ES 1 and inactive VSG genes in ES 3, 4, 5, 7, 11, 13 and 15. Relative interaction frequencies between the viewpoint and the SL-RNA locus on the right arm of chr. 9 is plotted. Bin size 20 kb. The analyses in a - b are based on Hi-C experiments with VSG-2 expressing cells (n=2, average interaction frequencies are shown). c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA locus marker – SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. The stacked bar graph depicts proportions of G1 or S phase nuclei with overlapping, adjacent or separate signals for the SL-RNA and VSG transcription compartments. Values are averages of three independent experiments and representative of two independent biological replicates (≥100 G1 or S phase nuclei); error bars, SD. Detailed n and p values are provided in Data S1 sheet 3. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices; scale bars 2 μm. N, nucleus; K, kinetoplast (mitochondrial genome).

    Techniques Used: Expressing, Stable Transfection, Hi-C, Immunofluorescence, Marker, Microscopy, Staining

    Pol I and tSNAP expression and localisation following knockdown of the VEX complex. a, Immunofluorescence-based analysis of VSG expression following tetracycline (Tet) inducible VEX1 knockdown, VEX2 knockdown or VEX1/VEX2 knockdown. VSG-2 (magenta) is the active- VSG and VSG-6 (green) is a silent- VSG in this strain. The stacked bar graph depicts percentages of VSG-2 single positive cells and VSG-2/VSG-6 double positive cells; values are averages of two independent experiments and two biological replicates. DNA was counter-stained with DAPI; scale bar 2 μm. b, Western-blot analysis of VEX2, Pol I, tSNAP myc , VSG-6 and VSG-2 expression following VEX1, VEX2 or VEX1/VEX2 knockdown. EF1α was used as a loading control. The data is representative of two independent experiments and two biological replicates. c-d , Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar and active- VSG marker (Pol I, large subunit). The stacked bar graph in c depicts proportions of G1 nuclei with tSNAP myc / Pol I overlapping, adjacent or separate signals following tetracycline (Tet) inducible VEX1 (48 h), VEX2 (12 h) or VEX1/VEX2 knockdown (12 h). tSNAP myc / active- VSG localisation were not monitored beyond 12 h following VEX2 and VEX1/2 knockdown as Pol I signal drops below detection at later time-points. The values are averages of two independent experiments and two biological replicates (≥100 G1 nuclei). In the box plot in d , the distance between the edges of the ESB and tSNAP foci was measured in > 81 G1 nuclei. The centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. In a/c , error bars, SD. In c-d , knockdown conditions were compared to parental cells using two-tailed paired ( c ) or unpaired ( d ) Student’s t -tests: *, p
    Figure Legend Snippet: Pol I and tSNAP expression and localisation following knockdown of the VEX complex. a, Immunofluorescence-based analysis of VSG expression following tetracycline (Tet) inducible VEX1 knockdown, VEX2 knockdown or VEX1/VEX2 knockdown. VSG-2 (magenta) is the active- VSG and VSG-6 (green) is a silent- VSG in this strain. The stacked bar graph depicts percentages of VSG-2 single positive cells and VSG-2/VSG-6 double positive cells; values are averages of two independent experiments and two biological replicates. DNA was counter-stained with DAPI; scale bar 2 μm. b, Western-blot analysis of VEX2, Pol I, tSNAP myc , VSG-6 and VSG-2 expression following VEX1, VEX2 or VEX1/VEX2 knockdown. EF1α was used as a loading control. The data is representative of two independent experiments and two biological replicates. c-d , Immunofluorescence-based colocalisation studies of tSNAP myc and a nucleolar and active- VSG marker (Pol I, large subunit). The stacked bar graph in c depicts proportions of G1 nuclei with tSNAP myc / Pol I overlapping, adjacent or separate signals following tetracycline (Tet) inducible VEX1 (48 h), VEX2 (12 h) or VEX1/VEX2 knockdown (12 h). tSNAP myc / active- VSG localisation were not monitored beyond 12 h following VEX2 and VEX1/2 knockdown as Pol I signal drops below detection at later time-points. The values are averages of two independent experiments and two biological replicates (≥100 G1 nuclei). In the box plot in d , the distance between the edges of the ESB and tSNAP foci was measured in > 81 G1 nuclei. The centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. In a/c , error bars, SD. In c-d , knockdown conditions were compared to parental cells using two-tailed paired ( c ) or unpaired ( d ) Student’s t -tests: *, p

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot, Marker, Two Tailed Test

    The exclusive association between the active VSG and the SL -locus is VEX2-dependent. a-b, Immunofluorescence and super resolution microscopy based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and Pol I (nucleolus and active-VSG transcription compartment) following VEX1, VEX2 and VEX1/VEX2 knockdown. a, Representative images, all nuclei are G1. On the right-hand side, two representative histograms depict the distribution of signal intensity across the distance indicated by the dashed lines. Images correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. b, The box plot depicts the distance between the active-VSG and the nearest SL-RNA transcription compartment (≥ 81 G1 nuclei) following VEX1, VEX2 and VEX1/VEX2 knockdown; the centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. The distances for each knockdown condition were compared to the parental cell line using a two-tailed unpaired Student’s t -test. c, Hi-C (virtual 4C) analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the VSG expression sites are shown before and after VEX2 knockdown. Each dot represents the average value for one expression site. Bin size 20 kb. d, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and VSG expression sites is shown. Bin size 20 kb. e, Virtual 4C analysis, viewpoint: EP1 (chr. 10) or GPEET gene array (chr. 6). Relative interaction frequencies between the viewpoint and the SL-RNA locus are plotted. Bin size 20 kb. The analyses in c to e are based on Hi-C experiments with cells before and 24 h after VEX2 knockdown (n=3, the average is shown). f, Schematic model for monogenic VSG expression. A strong inter-chromosomal interaction between the SL -array and the active VSG gene facilitates spatial integration of transcription and mRNA maturation. VEX1 and VEX2 are primarily SL - and active-VSG associated, respectively, and sustain monogenic VSG expression by excluding other VSGs. The VSG-SL organelle is reconfigured upon activation of a different VSG.
    Figure Legend Snippet: The exclusive association between the active VSG and the SL -locus is VEX2-dependent. a-b, Immunofluorescence and super resolution microscopy based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and Pol I (nucleolus and active-VSG transcription compartment) following VEX1, VEX2 and VEX1/VEX2 knockdown. a, Representative images, all nuclei are G1. On the right-hand side, two representative histograms depict the distribution of signal intensity across the distance indicated by the dashed lines. Images correspond to maximal 3D projections of stacks of 0.1 μm slices; DNA was counter-stained with DAPI; scale bars 2 μm. b, The box plot depicts the distance between the active-VSG and the nearest SL-RNA transcription compartment (≥ 81 G1 nuclei) following VEX1, VEX2 and VEX1/VEX2 knockdown; the centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend from maximal to the minimal values; all data points are shown. The distances for each knockdown condition were compared to the parental cell line using a two-tailed unpaired Student’s t -test. c, Hi-C (virtual 4C) analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the VSG expression sites are shown before and after VEX2 knockdown. Each dot represents the average value for one expression site. Bin size 20 kb. d, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and VSG expression sites is shown. Bin size 20 kb. e, Virtual 4C analysis, viewpoint: EP1 (chr. 10) or GPEET gene array (chr. 6). Relative interaction frequencies between the viewpoint and the SL-RNA locus are plotted. Bin size 20 kb. The analyses in c to e are based on Hi-C experiments with cells before and 24 h after VEX2 knockdown (n=3, the average is shown). f, Schematic model for monogenic VSG expression. A strong inter-chromosomal interaction between the SL -array and the active VSG gene facilitates spatial integration of transcription and mRNA maturation. VEX1 and VEX2 are primarily SL - and active-VSG associated, respectively, and sustain monogenic VSG expression by excluding other VSGs. The VSG-SL organelle is reconfigured upon activation of a different VSG.

    Techniques Used: Immunofluorescence, Microscopy, Staining, Two Tailed Test, Hi-C, Expressing, Activation Assay

    Genome-wide interaction frequencies of VSG genes in expression sites and the SL-RNA locus. a, Hi-C (virtual 4C) analysis with locations of viewpoints marked by pink boxes. Viewpoints VSG ES 4, 7, 11 and 17 are located on intermediate chromosomes that are not depicted in this figure. Interaction frequencies between each viewpoint and the 11 mega-base chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions of each chromosome are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)–3’(haplotype B). Bin size 50 kb. Virtual 4C analyses in a-b are based on Hi-C experiments of VSG-2 expressing cells (n=2, the average is shown). b, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the active VSG ES 1 (cyan) and inactive VSG ESs (magenta) is shown. Each dot represents the average value for one expression site. Bin size 20 kb. c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices and are representative of two biological replicates and three independent experiments; scale bars 2 μm.
    Figure Legend Snippet: Genome-wide interaction frequencies of VSG genes in expression sites and the SL-RNA locus. a, Hi-C (virtual 4C) analysis with locations of viewpoints marked by pink boxes. Viewpoints VSG ES 4, 7, 11 and 17 are located on intermediate chromosomes that are not depicted in this figure. Interaction frequencies between each viewpoint and the 11 mega-base chromosomes are shown. Chromosome cores, dark grey; sub-telomeric regions, light grey. The hemizygous sub-telomeric regions of each chromosome are displayed in the following order: 5’(haplotype A)–5’(haplotype B)–diploid chromosome core–3’(haplotype A)–3’(haplotype B). Bin size 50 kb. Virtual 4C analyses in a-b are based on Hi-C experiments of VSG-2 expressing cells (n=2, the average is shown). b, Virtual 4C analysis, viewpoint: SL-RNA locus (chr. 9). Relative interaction frequencies between the viewpoint and the active VSG ES 1 (cyan) and inactive VSG ESs (magenta) is shown. Each dot represents the average value for one expression site. Bin size 20 kb. c, Immunofluorescence-based colocalisation studies of tSNAP myc (SL-RNA transcription compartment) and a nucleolar and active- VSG transcription compartment marker (Pol I, largest subunit) using super resolution microscopy. DNA was counter-stained with DAPI; the images correspond to maximal 3D projections of stacks of 0.1 μm slices and are representative of two biological replicates and three independent experiments; scale bars 2 μm.

    Techniques Used: Genome Wide, Expressing, Hi-C, Immunofluorescence, Marker, Microscopy, Staining

    18) Product Images from "Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction"

    Article Title: Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2016.05.017

    Comparison effects of Mg 2+ concentration on the activity of polα-prim and the Klenow fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of hetero-DNA primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).
    Figure Legend Snippet: Comparison effects of Mg 2+ concentration on the activity of polα-prim and the Klenow fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of hetero-DNA primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).

    Techniques Used: Concentration Assay, Activity Assay, Sequencing, Binding Assay

    Comparison of the effects of Zn 2+ alone and in combination with Mg 2+ on DNA synthesis by polα-prim and the Klenow fragment (A) Extension of hetero-DNA primers annealed with heterogeneous 73b template by polα-prim (enzyme to primer/template ratio = 1:10) and (B) the Klenow fragment. (enzyme to primer/template ratio = 1:5). Lanes 2–9, polα-prim for one minute; Lanes 10–17, polα-prim for eight minutes; Lanes 18–25, the Klenow fragment for one minute; all with 100 µM dNTP at 35 °C. Reactions contained no enzyme (lane 1), no additional Me 2+ (lanes 2, 10, 18), 8 mM Mg 2+ (lanes 3, 11, 19), 10 to 50 µM Zn 2+ (lanes 4–6, 12–14, 20–22), and 8 mM Mg 2+ with 10 to 50 µM Zn 2+ (lanes 7–9, 15–17, 23–25).
    Figure Legend Snippet: Comparison of the effects of Zn 2+ alone and in combination with Mg 2+ on DNA synthesis by polα-prim and the Klenow fragment (A) Extension of hetero-DNA primers annealed with heterogeneous 73b template by polα-prim (enzyme to primer/template ratio = 1:10) and (B) the Klenow fragment. (enzyme to primer/template ratio = 1:5). Lanes 2–9, polα-prim for one minute; Lanes 10–17, polα-prim for eight minutes; Lanes 18–25, the Klenow fragment for one minute; all with 100 µM dNTP at 35 °C. Reactions contained no enzyme (lane 1), no additional Me 2+ (lanes 2, 10, 18), 8 mM Mg 2+ (lanes 3, 11, 19), 10 to 50 µM Zn 2+ (lanes 4–6, 12–14, 20–22), and 8 mM Mg 2+ with 10 to 50 µM Zn 2+ (lanes 7–9, 15–17, 23–25).

    Techniques Used: DNA Synthesis

    19) Product Images from "Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology"

    Article Title: Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq512

    Analysis of the overhang types created by ZFNs. ( A ) Scheme to determine ZFN overhangs. A supercoiled plasmid with a ZFN cleavage site is cut by a titration of in vitro transcribed and translated ZFNs. ZFN-linearized plasmids are purified by gel electrophoresis, 5′ overhangs filled in with Klenow polymerase (grey nucleotides), and the resulting blunt ends ligated. The mixture is subjected to high-throughput DNA sequencing. ( B ) Overhang types generated by a control restriction enzyme (HindIII) and three of the ZFN pairs used in this work. For clarity, only one DNA strand is shown. Enzyme binding sites are shown in grey; only the flanking three nucleotides are shown for ZFN binding sites. Primary cleavage sites, black triangles; secondary and tertiary cleavage sites, dark and light grey triangles, respectively; deletions, Δ. Microhomology within the target site can prevent unambiguous deduction of the overhang type. In such situations the possible overhangs are shown as joined triangles. Either of the two indicated thymidine residues may have been deleted after HindIII digestion.
    Figure Legend Snippet: Analysis of the overhang types created by ZFNs. ( A ) Scheme to determine ZFN overhangs. A supercoiled plasmid with a ZFN cleavage site is cut by a titration of in vitro transcribed and translated ZFNs. ZFN-linearized plasmids are purified by gel electrophoresis, 5′ overhangs filled in with Klenow polymerase (grey nucleotides), and the resulting blunt ends ligated. The mixture is subjected to high-throughput DNA sequencing. ( B ) Overhang types generated by a control restriction enzyme (HindIII) and three of the ZFN pairs used in this work. For clarity, only one DNA strand is shown. Enzyme binding sites are shown in grey; only the flanking three nucleotides are shown for ZFN binding sites. Primary cleavage sites, black triangles; secondary and tertiary cleavage sites, dark and light grey triangles, respectively; deletions, Δ. Microhomology within the target site can prevent unambiguous deduction of the overhang type. In such situations the possible overhangs are shown as joined triangles. Either of the two indicated thymidine residues may have been deleted after HindIII digestion.

    Techniques Used: Plasmid Preparation, Titration, In Vitro, Purification, Nucleic Acid Electrophoresis, High Throughput Screening Assay, DNA Sequencing, Generated, Binding Assay

    20) Product Images from "Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq"

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

    Journal: bioRxiv

    doi: 10.1101/2020.08.10.243931

    Visualisation of replication fork stalling sites by TrAEL-seq A : Potential processing pathways of a stalled replication fork. Lagging strand processing is likely to finish soon after stalling, and at least for the yeast RFB it is known that the lagging strand RNA primer is removed [ 51 ]. The fork could then undergo fork reversal to yield a Holliday junction or be cleaved on the leading or lagging strand. Whereas cleavage is irreversible and requires a recombination event to re-start the replication fork, reversed forks can revert to the normal replication fork structure by Holliday Junction migration (labelled HJ migration). The various 3’ DNA ends are marked with dots, either green for those predicted to be TrAEL-seq substrates or red for those that are unlikely to be tailed by TdT. The RNA primer on the Okazaki fragment in the leftmost structure is shown in green. B : Comparison of the yeast rDNA RFB signals in TrAEL-seq datasets compared to qDSB-seq (SRA accession: SRX5576747) [ 13 ] and GLOE-seq (SRA accessions: SRX6436839 and SRX6436840) [ 36 ]. Reads were quantified in 1 nucleotide steps and are shown as raw read counts. Note that the three TrAEL-seq datasets are shown on the same scale and that the total number of de-duplicated reads in these libraries is very similar (range 1.68-1.97×10 6 reads), so the absence of signal in fob1 Δ is due to the absence of replication fork stalling rather than lower library complexity. Scales were altered for qDSB-seq and GLOE-seq datasets because of varying sequence depth and signal-to-noise ratio. qDSB-seq data was obtained from S-phase synchronised cells, all other samples are from asynchronous log-phase cell populations growing in YPD media. Schematic diagram shows the positions of RFB elements previously mapped by 2D gel electrophoresis [ 45 , 46 ], and black triangles indicate previously mapped sites of DNA ends [ 47 , 51 ]. C : rDNA TrAEL-seq reads for undifferentiated and retinoic acid-treated hESCs, normalised so that both samples have equivalent total reads after deduplication. Reads were summed in 100 bp sliding windows spaced every 10 bp. One rDNA repeat is shown, the RNA polymerase I-transcribed 45S RNA is shown as a grey line with mature rRNAs marked in green in the schematic diagram. Note that the 45S gene is shown as transcribed right-to-left to maintain consistency with the yeast data. The R repeats, which contain the RFBs, are marked in green, while the primary direction of replication is shown by a red arrow labelled as ‘Replication?’ to take into account evidence that forks can move in both directions through the human rDNA. D : Average TrAEL-seq profiles across centromeres +/- 1kb for wild type (red), fob1 Δ (orange) and rad52 Δ (purple) cells. Centromeres are categorised based on replication direction in the yeast genome assembly into those replicated forward (CEN3, CEN5, CEN13), reverse (CEN11, CEN15, CEN10, CEN8, CEN12, CEN9) and those in termination zones that could be replicated in either direction (CEN14, CEN16, CEN1, CEN4, CEN7, CEN6, CEN2), see Fig. S2A for details. E: Average TrAEL-seq profiles across tRNAs +/- 200 bp for wild type, fob1 Δ and rad52 Δ cells. tRNAs are categorised into those for which transcription is co-directional with the replication fork and those for which transcription is head-on to the direction of the replication fork. tRNAs for which the replication direction is not well defined were excluded. Arrows indicate peaks that are dependent on replication direction.
    Figure Legend Snippet: Visualisation of replication fork stalling sites by TrAEL-seq A : Potential processing pathways of a stalled replication fork. Lagging strand processing is likely to finish soon after stalling, and at least for the yeast RFB it is known that the lagging strand RNA primer is removed [ 51 ]. The fork could then undergo fork reversal to yield a Holliday junction or be cleaved on the leading or lagging strand. Whereas cleavage is irreversible and requires a recombination event to re-start the replication fork, reversed forks can revert to the normal replication fork structure by Holliday Junction migration (labelled HJ migration). The various 3’ DNA ends are marked with dots, either green for those predicted to be TrAEL-seq substrates or red for those that are unlikely to be tailed by TdT. The RNA primer on the Okazaki fragment in the leftmost structure is shown in green. B : Comparison of the yeast rDNA RFB signals in TrAEL-seq datasets compared to qDSB-seq (SRA accession: SRX5576747) [ 13 ] and GLOE-seq (SRA accessions: SRX6436839 and SRX6436840) [ 36 ]. Reads were quantified in 1 nucleotide steps and are shown as raw read counts. Note that the three TrAEL-seq datasets are shown on the same scale and that the total number of de-duplicated reads in these libraries is very similar (range 1.68-1.97×10 6 reads), so the absence of signal in fob1 Δ is due to the absence of replication fork stalling rather than lower library complexity. Scales were altered for qDSB-seq and GLOE-seq datasets because of varying sequence depth and signal-to-noise ratio. qDSB-seq data was obtained from S-phase synchronised cells, all other samples are from asynchronous log-phase cell populations growing in YPD media. Schematic diagram shows the positions of RFB elements previously mapped by 2D gel electrophoresis [ 45 , 46 ], and black triangles indicate previously mapped sites of DNA ends [ 47 , 51 ]. C : rDNA TrAEL-seq reads for undifferentiated and retinoic acid-treated hESCs, normalised so that both samples have equivalent total reads after deduplication. Reads were summed in 100 bp sliding windows spaced every 10 bp. One rDNA repeat is shown, the RNA polymerase I-transcribed 45S RNA is shown as a grey line with mature rRNAs marked in green in the schematic diagram. Note that the 45S gene is shown as transcribed right-to-left to maintain consistency with the yeast data. The R repeats, which contain the RFBs, are marked in green, while the primary direction of replication is shown by a red arrow labelled as ‘Replication?’ to take into account evidence that forks can move in both directions through the human rDNA. D : Average TrAEL-seq profiles across centromeres +/- 1kb for wild type (red), fob1 Δ (orange) and rad52 Δ (purple) cells. Centromeres are categorised based on replication direction in the yeast genome assembly into those replicated forward (CEN3, CEN5, CEN13), reverse (CEN11, CEN15, CEN10, CEN8, CEN12, CEN9) and those in termination zones that could be replicated in either direction (CEN14, CEN16, CEN1, CEN4, CEN7, CEN6, CEN2), see Fig. S2A for details. E: Average TrAEL-seq profiles across tRNAs +/- 200 bp for wild type, fob1 Δ and rad52 Δ cells. tRNAs are categorised into those for which transcription is co-directional with the replication fork and those for which transcription is head-on to the direction of the replication fork. tRNAs for which the replication direction is not well defined were excluded. Arrows indicate peaks that are dependent on replication direction.

    Techniques Used: Migration, Sequencing, Two-Dimensional Gel Electrophoresis, Electrophoresis

    21) Product Images from "ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes"

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1249

    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
    Figure Legend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Techniques Used: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

    22) Product Images from "Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease"

    Article Title: Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-170342

    Fe-dependent ROS generation and α-Syn aggregation in pCW-iFLAG-α-Syn SHSY-5Y cells. A, B) Cells treated with FeSO 4 but not CuSO 4 showed a significant increase in nuclear and mitochondrial ROS load. Results are presented as percentage of ROS-positive cells; error bars in the histogram represent the standard error of the mean (SEM) from three independent experiments. C) Immunofluorescence indicates α-Syn aggregate formation in FeSO 4 -treated cells. ** p ≤ 0.01.
    Figure Legend Snippet: Fe-dependent ROS generation and α-Syn aggregation in pCW-iFLAG-α-Syn SHSY-5Y cells. A, B) Cells treated with FeSO 4 but not CuSO 4 showed a significant increase in nuclear and mitochondrial ROS load. Results are presented as percentage of ROS-positive cells; error bars in the histogram represent the standard error of the mean (SEM) from three independent experiments. C) Immunofluorescence indicates α-Syn aggregate formation in FeSO 4 -treated cells. ** p ≤ 0.01.

    Techniques Used: Immunofluorescence

    α-Syn DNA nicking activity may be mediated by its oxidation. A) Exposure of recombinant α-Syn to 1 O 2 , generated by exposing flavonoid Bengal red to UVB radiation, enhanced its DNA nicking activity with plasmid scDNA in vitro . The products were analyzed by agarose gel electrophoresis. The histogram represents quantification of DNA fragmentation. B) SHSY-5Y cells were exposed to similarly oxidized α-Syn, and DNA damage was quantified by alkaline Comet assay.
    Figure Legend Snippet: α-Syn DNA nicking activity may be mediated by its oxidation. A) Exposure of recombinant α-Syn to 1 O 2 , generated by exposing flavonoid Bengal red to UVB radiation, enhanced its DNA nicking activity with plasmid scDNA in vitro . The products were analyzed by agarose gel electrophoresis. The histogram represents quantification of DNA fragmentation. B) SHSY-5Y cells were exposed to similarly oxidized α-Syn, and DNA damage was quantified by alkaline Comet assay.

    Techniques Used: Activity Assay, Recombinant, Generated, Plasmid Preparation, In Vitro, Agarose Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis

    Increased α-Syn expression causes DNA breaks in neurons synergistically with pro-oxidant metals. A, B) Alkaline Comet assay in iFLAG-α-Syn SHSY-5Y cells exposed to 200 μM FeSO 4 or CuSO 4 . α-Syn was induced with Dox for 48 h. Metal salts alone at the same concentration caused only moderate increases in strand breaks. C, D) Semi-quantitative LA-PCR assay for genomic DNA isolated from pCW-iFLAG-α-Syn SHSY-5Y cells in the presence of FeSO 4 or CuSO 4 . *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Increased α-Syn expression causes DNA breaks in neurons synergistically with pro-oxidant metals. A, B) Alkaline Comet assay in iFLAG-α-Syn SHSY-5Y cells exposed to 200 μM FeSO 4 or CuSO 4 . α-Syn was induced with Dox for 48 h. Metal salts alone at the same concentration caused only moderate increases in strand breaks. C, D) Semi-quantitative LA-PCR assay for genomic DNA isolated from pCW-iFLAG-α-Syn SHSY-5Y cells in the presence of FeSO 4 or CuSO 4 . *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Expressing, Alkaline Single Cell Gel Electrophoresis, Concentration Assay, Polymerase Chain Reaction, Isolation

    α-Syn and Fe synergistically induce neuronal apoptosis. A) pCW-iFLAG-α-Syn SHSY-5Y cells were induced with Dox for 48 h and then treated with 200 μM FeSO 4 or CuSO 4 for 24 h before being double-stained with Annexin V/PI and analyzed by flow cytometry. B)Results are presented as percentage of total apoptotic cells (early apoptotic (Q3)+ late apoptotic (Q2)). Error bars represent the SEM from three independent experiments. Fe or Cu alone caused a 4–8% necrosis (Q4) in uninduced cells, which was prevented by α-Syn induced cells. * p ≤ 0.05; ** p ≤ 0.01.
    Figure Legend Snippet: α-Syn and Fe synergistically induce neuronal apoptosis. A) pCW-iFLAG-α-Syn SHSY-5Y cells were induced with Dox for 48 h and then treated with 200 μM FeSO 4 or CuSO 4 for 24 h before being double-stained with Annexin V/PI and analyzed by flow cytometry. B)Results are presented as percentage of total apoptotic cells (early apoptotic (Q3)+ late apoptotic (Q2)). Error bars represent the SEM from three independent experiments. Fe or Cu alone caused a 4–8% necrosis (Q4) in uninduced cells, which was prevented by α-Syn induced cells. * p ≤ 0.05; ** p ≤ 0.01.

    Techniques Used: Staining, Flow Cytometry, Cytometry

    MD simulation: α-Syn N-terminal residues may be involved in DNA binding. Protein-DNA docking Model 1 demonstrating binding of α-Syn (PDB: 1XQ8) N-terminal amino acid residues Glu-35, Ser-42, Thr-54 to the crystal structure of d(CCGGTACCGG) as a B-DNA duplex (PDB: 3IXN). This structure model represents the best structure from the biggest cluster after the refinement process. Structures were analyzed using PyMOL Molecular Graphics System, Version 1.7.4.5 Schrödinger, LLC.
    Figure Legend Snippet: MD simulation: α-Syn N-terminal residues may be involved in DNA binding. Protein-DNA docking Model 1 demonstrating binding of α-Syn (PDB: 1XQ8) N-terminal amino acid residues Glu-35, Ser-42, Thr-54 to the crystal structure of d(CCGGTACCGG) as a B-DNA duplex (PDB: 3IXN). This structure model represents the best structure from the biggest cluster after the refinement process. Structures were analyzed using PyMOL Molecular Graphics System, Version 1.7.4.5 Schrödinger, LLC.

    Techniques Used: Binding Assay

    Model illustrating how α-Syn-induced DNA breaks contributes to neuronal apoptosis in PD. The role of pro-oxidant Fe or ROS in promoting α-Syn misfolding and oxidation, which could exacerbate its DNA nicking activity.
    Figure Legend Snippet: Model illustrating how α-Syn-induced DNA breaks contributes to neuronal apoptosis in PD. The role of pro-oxidant Fe or ROS in promoting α-Syn misfolding and oxidation, which could exacerbate its DNA nicking activity.

    Techniques Used: Activity Assay

    Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.
    Figure Legend Snippet: Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.

    Techniques Used: Binding Assay, Stable Transfection, Plasmid Preparation, Immunofluorescence, Proximity Ligation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, In Vitro, Co-Elution Assay, Recombinant, Labeling

    Nuclear α-Syn is required for inducing genome damage in neurons. A) Immunofluorescence images demonstrating nucleus- or cytoplasm-specific expression of mutant α-Syn vectors containing additional NES or NLS after their transient transfection in SHSY-5Y cells. B) Immunoblotting of cytosolic and nuclear fractions from WT-, NES-, and NLS-α-Syn-expressing SHSY-5Y cells. C, D) Alkaline Comet assay in NLS-, NES-, or WT-α-Syn-expressing cells with or without FeSO 4 or CuSO 4 .
    Figure Legend Snippet: Nuclear α-Syn is required for inducing genome damage in neurons. A) Immunofluorescence images demonstrating nucleus- or cytoplasm-specific expression of mutant α-Syn vectors containing additional NES or NLS after their transient transfection in SHSY-5Y cells. B) Immunoblotting of cytosolic and nuclear fractions from WT-, NES-, and NLS-α-Syn-expressing SHSY-5Y cells. C, D) Alkaline Comet assay in NLS-, NES-, or WT-α-Syn-expressing cells with or without FeSO 4 or CuSO 4 .

    Techniques Used: Immunofluorescence, Expressing, Mutagenesis, Transfection, Alkaline Single Cell Gel Electrophoresis

    Recombinant α-Syn nicks naked DNA in vitro , which is enhanced by its misfolding/oligomerization. A) Agarose gel electrophoresis showing plasmid scDNA cleavage by recombinant α-Syn. B) Assessment of SSBs and DSBs in scDNA induced by α-Syn. Values are expressed as SSBs and DSBs induced per μg of scDNA. C) Recombinant α-Syn was incubated with constant stirring to cause its aggregation, which was monitored by Thio T fluorescence analysis with an aliquot of α-Syn taken at various time intervals. D) The α-Syn aliquots were also analyzed for their DNA nicking activity with plasmid DNA by agarose gel electrophoresis. E) DNA breaks were quantified. F) Misfolding/β-sheet formation in α-Syn upon stirring as confirmed by CD spectroscopy.
    Figure Legend Snippet: Recombinant α-Syn nicks naked DNA in vitro , which is enhanced by its misfolding/oligomerization. A) Agarose gel electrophoresis showing plasmid scDNA cleavage by recombinant α-Syn. B) Assessment of SSBs and DSBs in scDNA induced by α-Syn. Values are expressed as SSBs and DSBs induced per μg of scDNA. C) Recombinant α-Syn was incubated with constant stirring to cause its aggregation, which was monitored by Thio T fluorescence analysis with an aliquot of α-Syn taken at various time intervals. D) The α-Syn aliquots were also analyzed for their DNA nicking activity with plasmid DNA by agarose gel electrophoresis. E) DNA breaks were quantified. F) Misfolding/β-sheet formation in α-Syn upon stirring as confirmed by CD spectroscopy.

    Techniques Used: Recombinant, In Vitro, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Fluorescence, Activity Assay, Spectroscopy

    DNA damage in neurons generated from normal and PD patient-derived SNCA -tri iPSC cells. A) Phase contrast image demonstrating generation of NPCs from iPSCs: (a) SNCA- tri iPSCs cultured in MEF feeder layer, (b) SNCA- tri iPSCs cultured in feeder free layer, (c) day 2, (d) day 4, (e) day 6 of neural induction for NPC derivation, (f) NPC at passage 3. The iPSC specific marker Oct4 and neural precursor markers nestin analyzed by immunoblotting (B). C) Immunofluorescence characterization with nestin and α-Syn protein expression. D) α-SYN mRNA quantitation in control versus SNCA-tri iPSC and NPC cells. E, F) LA-PCR analysis of genomic DNA isolated from control or SNCA -tri NPC cells exposed to 200 μM FeSO 4 or CuSO 4 . *** p ≤ 0.001.
    Figure Legend Snippet: DNA damage in neurons generated from normal and PD patient-derived SNCA -tri iPSC cells. A) Phase contrast image demonstrating generation of NPCs from iPSCs: (a) SNCA- tri iPSCs cultured in MEF feeder layer, (b) SNCA- tri iPSCs cultured in feeder free layer, (c) day 2, (d) day 4, (e) day 6 of neural induction for NPC derivation, (f) NPC at passage 3. The iPSC specific marker Oct4 and neural precursor markers nestin analyzed by immunoblotting (B). C) Immunofluorescence characterization with nestin and α-Syn protein expression. D) α-SYN mRNA quantitation in control versus SNCA-tri iPSC and NPC cells. E, F) LA-PCR analysis of genomic DNA isolated from control or SNCA -tri NPC cells exposed to 200 μM FeSO 4 or CuSO 4 . *** p ≤ 0.001.

    Techniques Used: Generated, Derivative Assay, Cell Culture, Marker, Immunofluorescence, Expressing, Quantitation Assay, Polymerase Chain Reaction, Isolation

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    Incubation:

    Article Title: Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes
    Article Snippet: .. For end-repair and biotin removal from un-ligated ends, 70 μl of end-repair mix was added (1× Ligation buffer (NEB), 357 μM dNTPs, 25U T4 PNK (NEB, M0201), 7.5U T4 DNA polymerase I (NEB, M0203), 2.5U DNA polymerase I, large (Klenow) fragment (NEB, M0210)) and incubated for 30 min at 20 °C and 20 min at 75 °C. .. To inactivate the enzymes, EDTA was added to a final concentration of 10 mM.

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer
    Article Snippet: .. DNA smears (1 μg) were incubated with dTTP, dATP, fluorescein–12-dCTP (catalog number NEL434001EA; PerkinElmer), fluorescein–12-dGTP (catalog number NEL496001EA; PerkinElmer), and the E. coli polymerase I Klenow fragment (catalog number M0210S; NEB) at 37°C for 30 min. Purified DNA was run on a 6% TBE-polyacrylamide gel; labeling was visualized using a Typhoon 9400 laser scanner (excitation wavelength, 488 nm; emission wavelength, 520 nm; GE Healthcare Life Sciences). .. Total DNA was visualized by UV after EtBr staining.

    other:

    Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents
    Article Snippet: MaterialsThe exo I and Klenow DNA polymerase were purchased from New England Biolab (Ipswich, MA).

    Article Title: Construction of a circular single-stranded DNA template containing a defined lesion
    Article Snippet: E.coli DNA polymerase I (Klenow Fragment) [pol I (Kf)], E.coli Exonuclease III, E.coli Exonuclease I, E.coli Uracil DNA glycosylase, E.coli RecA, T7 DNA polymerase, T4 Polynucleotide kinase, and M13KO7 helper phage were all purchased from New England Biolabs (Ipswich, MA).

    Ligation:

    Article Title: Antigenic variation by switching inter-chromosomal interactions with an RNA splicing locus in trypanosomes
    Article Snippet: .. For end-repair and biotin removal from un-ligated ends, 70 μl of end-repair mix was added (1× Ligation buffer (NEB), 357 μM dNTPs, 25U T4 PNK (NEB, M0201), 7.5U T4 DNA polymerase I (NEB, M0203), 2.5U DNA polymerase I, large (Klenow) fragment (NEB, M0210)) and incubated for 30 min at 20 °C and 20 min at 75 °C. .. To inactivate the enzymes, EDTA was added to a final concentration of 10 mM.

    Labeling:

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer
    Article Snippet: .. DNA smears (1 μg) were incubated with dTTP, dATP, fluorescein–12-dCTP (catalog number NEL434001EA; PerkinElmer), fluorescein–12-dGTP (catalog number NEL496001EA; PerkinElmer), and the E. coli polymerase I Klenow fragment (catalog number M0210S; NEB) at 37°C for 30 min. Purified DNA was run on a 6% TBE-polyacrylamide gel; labeling was visualized using a Typhoon 9400 laser scanner (excitation wavelength, 488 nm; emission wavelength, 520 nm; GE Healthcare Life Sciences). .. Total DNA was visualized by UV after EtBr staining.

    Purification:

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer
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    New England Biolabs klenow dna polymerase
    The principle of PE-PCR for bacterial <t>DNA</t> amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and <t>Klenow</t> DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).
    Klenow Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Journal: PLoS ONE

    Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    doi: 10.1371/journal.pone.0020303

    Figure Lengend Snippet: The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Article Snippet: Materials The exo I and Klenow DNA polymerase were purchased from New England Biolab (Ipswich, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Incubation, BAC Assay

    Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Journal: Nature

    Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

    doi: 10.1038/nature25449

    Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

    Techniques: Inhibition, Immunofluorescence, Staining

    Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template

    Journal:

    Article Title: Effect of Cross-Link Structure on DNA Interstrand Cross-Link Repair Synthesis

    doi: 10.1021/tx9000896

    Figure Lengend Snippet: Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template

    Article Snippet: Protected deoxyribonucleoside-3′-O-methylphosphonamidites were a product of JBL, Inc. Polynucleotide kinase, T4 DNA ligase, E. coli DNA polymerase I (Klenow fragment), and T7 DNA polymerase were obtained from New England Biolabs, Inc. Shrimp alkaline phosphatase (SAP) was from Roche Diagnostics.

    Techniques: Primer Extension Assay

    Pol ε poorly extends D-loops in comparison to Pol δ but is proficient to extend primed single-stranded DNA using the same substrates under the same conditions. ( A ) In vitro D-loop reactions using a 37-mer oligonucleotide were reconstituted using purified S. cerevisiae proteins as described in Materials and Methods. ( B ) Product analysis of reconstituted D-loop reactions containing either Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) at 0, 2 (not for Klenow), 5 and 10 min extension times. ( C ) Extension of primed single-stranded circular template DNA using a 37-mer oligonucleotide. ( D ) Product analysis of primer extension on denaturing gels of reaction containing Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) each plus or minus 10 nM PCNA/RFC at 0, 2 (not for Klenow), 5 and 10 min extension times. A 100 nt size ladder is shown in the left-most lane.

    Journal: Nucleic Acids Research

    Article Title: Strand displacement synthesis by yeast DNA polymerase ε

    doi: 10.1093/nar/gkw556

    Figure Lengend Snippet: Pol ε poorly extends D-loops in comparison to Pol δ but is proficient to extend primed single-stranded DNA using the same substrates under the same conditions. ( A ) In vitro D-loop reactions using a 37-mer oligonucleotide were reconstituted using purified S. cerevisiae proteins as described in Materials and Methods. ( B ) Product analysis of reconstituted D-loop reactions containing either Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) at 0, 2 (not for Klenow), 5 and 10 min extension times. ( C ) Extension of primed single-stranded circular template DNA using a 37-mer oligonucleotide. ( D ) Product analysis of primer extension on denaturing gels of reaction containing Klenow polymerase, Pol δ (10 nM) or Pol ε (10 nM) each plus or minus 10 nM PCNA/RFC at 0, 2 (not for Klenow), 5 and 10 min extension times. A 100 nt size ladder is shown in the left-most lane.

    Article Snippet: Klenow DNA polymerase was purchased from New England Biolabs.

    Techniques: In Vitro, Purification