m13mp18 Search Results


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  • 99
    New England Biolabs single stranded m13mp18 dna
    <t>DNA-dependent</t> DNA polymerase activity of subtype CRF02_AG WT and MR-RTs. Reactions were carried out at 37°C for the times indicated on top of the gel with 30 nM [ 32 P]-P 17 <t>/M13mp18,</t> 200 μM of each dNTP and 83 nM WT or MR-RT, or without RT (control) in a reaction volume of 10 μl. Extension products were analyzed by denaturing gel electrophoresis on a 10% sequencing gel and visualized by a phosphoimaging device. DNA size markers are shown on the left.
    Single Stranded M13mp18 Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs m13mp18 ssdna
    RPA Levels Differentially Affect Lagging- and Leading-Strand Priming (A) Primase assay. RPA was pre-bound to unprimed (left) and primed (right) <t>M13mp18</t> <t>ssDNA</t> for 10 min before addition of Pol α for 20 min. 120 nM RPA is saturating assuming a binding footprint of 30 nt. (B and C) Two-dimensional gels of replication assays performed on the 3 kb CPD LEAD template with 10 nM (B) or 100 nM (C) RPA. Lane profiles showing the constituents (denaturing) of the full-length products are shown below each gel. (D) RPA titration on an AhdI-linearized undamaged template. (E) RPA titration on a truncated undamaged template as illustrated.
    M13mp18 Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bayou Biolabs m13mp18 single stranded dna
    In vitro <t>DNA</t> repair synthesis and excision activity in extracts from G0/G1- and S-phase-arrested HCT-116 cells. ( a ) DNA repair synthesis on ε A-containing <t>M13mp18</t> DNA. The cell-free nuclear extracts (0–60 μg per reaction) were incubated
    M13mp18 Single Stranded Dna, supplied by Bayou Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Boehringer Mannheim m13mp18
    In vitro <t>DNA</t> repair synthesis and excision activity in extracts from G0/G1- and S-phase-arrested HCT-116 cells. ( a ) DNA repair synthesis on ε A-containing <t>M13mp18</t> DNA. The cell-free nuclear extracts (0–60 μg per reaction) were incubated
    M13mp18, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher m13mp18
    In vitro <t>DNA</t> repair synthesis and excision activity in extracts from G0/G1- and S-phase-arrested HCT-116 cells. ( a ) DNA repair synthesis on ε A-containing <t>M13mp18</t> DNA. The cell-free nuclear extracts (0–60 μg per reaction) were incubated
    M13mp18, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Bayou Biolabs m13mp18 plasmid
    In vitro <t>DNA</t> repair synthesis and excision activity in extracts from G0/G1- and S-phase-arrested HCT-116 cells. ( a ) DNA repair synthesis on ε A-containing <t>M13mp18</t> DNA. The cell-free nuclear extracts (0–60 μg per reaction) were incubated
    M13mp18 Plasmid, supplied by Bayou Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bayou Biolabs m13mp18 dsdna plasmids
    Abf2p truncations and their effect on DNA binding. ( A ) Schematic representation of the Abf2p deletion mutants. The domains are designated as in the text except for the linker, indicated with an ‘L’. Dashed lines indicate deleted regions. ( B ) Titration of Abf2p and Mut1 to Mut6 constructs binding on linearized <t>M13mp18</t> <t>dsDNA</t> (8 fmol) at 25°C, by EMSA. The protein/bp ratios are indicated and correspond respectively to 0.37, 0.75, 1.5, 3, 6 and 12 pmol of Abf2p. ( C ) Titration of Abf2p and Mut1 to Mut6 binding to four-way junction DNA (100 nM) by EMSA. ( D ) Effect of Abf2p truncations in vivo . Spot assay of cells with 1/5 serial dilutions on plate containing glycerol as carbon-source (‘YPG plate’) shows the effect of different Abf2p truncations on the ability to maintain y-mtDNA and thus on capability of yeast ( Saccharomyces cerevisiae ) cells to use non-fermentable carbon source. Full-length Abf2p and Mut6 (Abf2p without HMG-box 2) are capable of protecting mtDNA whereas Mut2 (Abf2p without N-flag and N-helix), Mut3 (N-helix+HMG-box 1) and Mut5 (HMG-box 2 alone) are not functional. ( E ) Visualization of mitochondrial nucleoids in cells harboring different Abf2p truncations by DAPI staining of DNA and fluorescence confocal microscopy. Abf2p and Mut6 maintain mtDNA whereas Mut2, Mut3 and Mut5 cannot. C (+) corresponds to wild-type haploids, and C (-) to Abf2 Δcells. The scale bars correspond to 2.5 μm.
    M13mp18 Dsdna Plasmids, supplied by Bayou Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa m13mp18 rf
    Chain-formation activity of HLTF with the multiply primed <t>M13mp18</t> ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.
    M13mp18 Rf, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Staples m13mp18 scaffold
    Chain-formation activity of HLTF with the multiply primed <t>M13mp18</t> ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.
    M13mp18 Scaffold, supplied by Staples, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Bayou Biolabs m13mp18 single stranded plasmid dna
    Chain-formation activity of HLTF with the multiply primed <t>M13mp18</t> ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.
    M13mp18 Single Stranded Plasmid Dna, supplied by Bayou Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA-dependent DNA polymerase activity of subtype CRF02_AG WT and MR-RTs. Reactions were carried out at 37°C for the times indicated on top of the gel with 30 nM [ 32 P]-P 17 /M13mp18, 200 μM of each dNTP and 83 nM WT or MR-RT, or without RT (control) in a reaction volume of 10 μl. Extension products were analyzed by denaturing gel electrophoresis on a 10% sequencing gel and visualized by a phosphoimaging device. DNA size markers are shown on the left.

    Journal: Nucleic Acids Research

    Article Title: Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

    doi: 10.1093/nar/gkw060

    Figure Lengend Snippet: DNA-dependent DNA polymerase activity of subtype CRF02_AG WT and MR-RTs. Reactions were carried out at 37°C for the times indicated on top of the gel with 30 nM [ 32 P]-P 17 /M13mp18, 200 μM of each dNTP and 83 nM WT or MR-RT, or without RT (control) in a reaction volume of 10 μl. Extension products were analyzed by denaturing gel electrophoresis on a 10% sequencing gel and visualized by a phosphoimaging device. DNA size markers are shown on the left.

    Article Snippet: DNA-dependent DNA polymerase activity assay A total of 100 pmol of 5′ [32 P]-end-labeled 17-mer DNA primer ( ) were annealed to single stranded M13mp18 DNA (New England BioLabs, Frankfurt, Germany).

    Techniques: Activity Assay, Nucleic Acid Electrophoresis, Sequencing

    DNA-dependent DNA polymerase activity of FV RT and HIV-1 RT. A 5′-end-labeled DNA primer was annealed to a single-stranded M13mp18 DNA template and extended with either HIV-1 RT or FV RT for the lengths of time indicated. Samples were precipitated with ethanol and then fractionated on a 2.0% alkaline agarose gel. After electrophoresis, the gel was neutralized and dried as described in Materials and Methods. The products were visualized by autoradiography. The size marker was 5′-end-labeled 1.0-kb marker from New England Biolabs.

    Journal: Journal of Virology

    Article Title: Characterization of the Polymerase and RNase H Activities of Human Foamy Virus Reverse Transcriptase

    doi: 10.1128/JVI.78.12.6112-6121.2004

    Figure Lengend Snippet: DNA-dependent DNA polymerase activity of FV RT and HIV-1 RT. A 5′-end-labeled DNA primer was annealed to a single-stranded M13mp18 DNA template and extended with either HIV-1 RT or FV RT for the lengths of time indicated. Samples were precipitated with ethanol and then fractionated on a 2.0% alkaline agarose gel. After electrophoresis, the gel was neutralized and dried as described in Materials and Methods. The products were visualized by autoradiography. The size marker was 5′-end-labeled 1.0-kb marker from New England Biolabs.

    Article Snippet: The primer −47 (New England Biolabs) was 5′-end labeled and then annealed to single-strand M13mp18 DNA (New England Biolabs).

    Techniques: Activity Assay, Labeling, Agarose Gel Electrophoresis, Electrophoresis, Autoradiography, Marker

    Detection of viral RNA using DNA nanoswitches.

    Journal: bioRxiv

    Article Title: Programmable low-cost DNA-based platform for viral RNA detection

    doi: 10.1101/2020.01.12.902452

    Figure Lengend Snippet: Detection of viral RNA using DNA nanoswitches.

    Article Snippet: Nanoswitches were constructed as described previously., A genomic single-stranded DNA (New England Biolabs M13mp18) was linearized using targeted cleavage with BtsCI restriction enzyme.

    Techniques:

    DNA nanoswitches specifically and differentially detect RNA from two different flaviviruses and between two highly similar ZIKV isolates. (a) ZIKV nanoswitches specifically detect ZIKV RNA but not DENV RNA, and vice versa . ( b) Multiplexed detection of ZIKV and DENV RNA. ( c) Illustration showing culture a nd RNA extraction of ZIKV Cambodia and Uganda strains. The mismatches in a representative target sequen ce between the two strains are shown. ( d) Specificity test of Cambodia and Uganda strains of ZIKV RNA. * denotes a band of contaminating cellular DNA following RNA isolation.

    Journal: bioRxiv

    Article Title: Programmable low-cost DNA-based platform for viral RNA detection

    doi: 10.1101/2020.01.12.902452

    Figure Lengend Snippet: DNA nanoswitches specifically and differentially detect RNA from two different flaviviruses and between two highly similar ZIKV isolates. (a) ZIKV nanoswitches specifically detect ZIKV RNA but not DENV RNA, and vice versa . ( b) Multiplexed detection of ZIKV and DENV RNA. ( c) Illustration showing culture a nd RNA extraction of ZIKV Cambodia and Uganda strains. The mismatches in a representative target sequen ce between the two strains are shown. ( d) Specificity test of Cambodia and Uganda strains of ZIKV RNA. * denotes a band of contaminating cellular DNA following RNA isolation.

    Article Snippet: Nanoswitches were constructed as described previously., A genomic single-stranded DNA (New England Biolabs M13mp18) was linearized using targeted cleavage with BtsCI restriction enzyme.

    Techniques: RNA Extraction, Isolation

    Detection of viral RNA using DNA nanoswitches. ( a) Schematic of the fragmentation of viral RNA and subsequent detection by the DNA nanoswitch. (b) Fragmentation analysis of ZIKV RNA that was fragmented at 94 °C for 1, 3, 6, and 9 minutes. ( c) Proof-of-concept showing detection of a target region chosen from the literature. 28 ( d) Schematic of the design of multiple nanoswitches for detection with the signal multiplication strategy. ( e) Validation of the signal multiplication strategy: the detection signal was increased for a fixed pool of DNA targets when using multiple targeting nanoswitches. ( f) Detection sensitivity of the pooled nanoswitches for ZIKV RNA. Error bars represent standard deviation from triplicate experiments.

    Journal: bioRxiv

    Article Title: Programmable low-cost DNA-based platform for viral RNA detection

    doi: 10.1101/2020.01.12.902452

    Figure Lengend Snippet: Detection of viral RNA using DNA nanoswitches. ( a) Schematic of the fragmentation of viral RNA and subsequent detection by the DNA nanoswitch. (b) Fragmentation analysis of ZIKV RNA that was fragmented at 94 °C for 1, 3, 6, and 9 minutes. ( c) Proof-of-concept showing detection of a target region chosen from the literature. 28 ( d) Schematic of the design of multiple nanoswitches for detection with the signal multiplication strategy. ( e) Validation of the signal multiplication strategy: the detection signal was increased for a fixed pool of DNA targets when using multiple targeting nanoswitches. ( f) Detection sensitivity of the pooled nanoswitches for ZIKV RNA. Error bars represent standard deviation from triplicate experiments.

    Article Snippet: Nanoswitches were constructed as described previously., A genomic single-stranded DNA (New England Biolabs M13mp18) was linearized using targeted cleavage with BtsCI restriction enzyme.

    Techniques: Standard Deviation

    Prior extraction or pre-amplification of target RNA facilitates detection of ZIKV and SARS-CoV-2 RNA at clinically relevant levels in biofluids. (a) Positive identification of ZIKV RNA in spiked urine by first isolating in vitro transcribed target RNA using a commercially available viral RNA extraction kit, followed by direct, non-enzymatic detection using DNA nanoswitches. ( b) Positive identification of ZIKV RNA from virus particles spiked into urine based on NASBA. (c) Positive detection of in vitro transcribed SARS-CoV-2 RNA in human saliva based on NASBA. Error bars represent standard deviation from triplicate experiments.

    Journal: bioRxiv

    Article Title: Programmable low-cost DNA-based platform for viral RNA detection

    doi: 10.1101/2020.01.12.902452

    Figure Lengend Snippet: Prior extraction or pre-amplification of target RNA facilitates detection of ZIKV and SARS-CoV-2 RNA at clinically relevant levels in biofluids. (a) Positive identification of ZIKV RNA in spiked urine by first isolating in vitro transcribed target RNA using a commercially available viral RNA extraction kit, followed by direct, non-enzymatic detection using DNA nanoswitches. ( b) Positive identification of ZIKV RNA from virus particles spiked into urine based on NASBA. (c) Positive detection of in vitro transcribed SARS-CoV-2 RNA in human saliva based on NASBA. Error bars represent standard deviation from triplicate experiments.

    Article Snippet: Nanoswitches were constructed as described previously., A genomic single-stranded DNA (New England Biolabs M13mp18) was linearized using targeted cleavage with BtsCI restriction enzyme.

    Techniques: Amplification, In Vitro, RNA Extraction, Standard Deviation

    DNA nanoswitches directly detect ZIKV RNA from infected human liver cells. (a) RNA isolated from mock-infected Huh7 cells at 1, 2, and 3 days post infection show no ZIKV detection. ( b) RNA isolated from Zika-infected Huh7 cells at 1, 2, and 3 days post infection shows increasing ZIKV detection over time, with red arrows denoting detection bands. * denotes a band of contaminating cellular DNA following RNA extraction. ( c) Quantification of nanoswitch detection signal, with error bars representing standard deviation from triplicate experiments.

    Journal: bioRxiv

    Article Title: Programmable low-cost DNA-based platform for viral RNA detection

    doi: 10.1101/2020.01.12.902452

    Figure Lengend Snippet: DNA nanoswitches directly detect ZIKV RNA from infected human liver cells. (a) RNA isolated from mock-infected Huh7 cells at 1, 2, and 3 days post infection show no ZIKV detection. ( b) RNA isolated from Zika-infected Huh7 cells at 1, 2, and 3 days post infection shows increasing ZIKV detection over time, with red arrows denoting detection bands. * denotes a band of contaminating cellular DNA following RNA extraction. ( c) Quantification of nanoswitch detection signal, with error bars representing standard deviation from triplicate experiments.

    Article Snippet: Nanoswitches were constructed as described previously., A genomic single-stranded DNA (New England Biolabs M13mp18) was linearized using targeted cleavage with BtsCI restriction enzyme.

    Techniques: Infection, Isolation, RNA Extraction, Standard Deviation

    VP1 protein binds to various DNA molecules. Purified GST and GST-fused proteins were used for analysing the interaction of recombinant proteins with various DNA samples, such as linear dsDNA ( a ), minus-strand ssDNA ( b ), and M13mp18 phage DNA ( c ). All DNA samples were generated by different preparations as described in the Materials and Methods. After the agarose gel shift assay, the DNA fragment signals were observed by EtBr staining. The 1% SDS (underline lane-labelled 1% SDS) was also used to confirm the retardation caused by tested proteins. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and DNA molecules. The “pcDNA3.1 x Eco R I” indicated generation of the linear form of pcDNA3.1 DNA digested by Eco R I

    Journal: BMC Veterinary Research

    Article Title: Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus

    doi: 10.1186/s12917-018-1465-5

    Figure Lengend Snippet: VP1 protein binds to various DNA molecules. Purified GST and GST-fused proteins were used for analysing the interaction of recombinant proteins with various DNA samples, such as linear dsDNA ( a ), minus-strand ssDNA ( b ), and M13mp18 phage DNA ( c ). All DNA samples were generated by different preparations as described in the Materials and Methods. After the agarose gel shift assay, the DNA fragment signals were observed by EtBr staining. The 1% SDS (underline lane-labelled 1% SDS) was also used to confirm the retardation caused by tested proteins. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and DNA molecules. The “pcDNA3.1 x Eco R I” indicated generation of the linear form of pcDNA3.1 DNA digested by Eco R I

    Article Snippet: Pure M13mp18 single-stranded DNA along with circular ssDNA materials were purchased from New England BioLabs (#N4040S, NEB, USA).

    Techniques: Purification, Recombinant, Generated, Agarose Gel Electrophoresis, Shift Assay, Staining, Marker

    RPA ties NS5BΔ21 to the template. 35 S-labeled NS5BΔ21 and NS3h, individually synthesized in transcription/translation reactions in vitro in the presence of [ 35 S]methionine, were incubated with circular ssM13mp18 DNA coated with RPA (A and D) or gp32 (B and E) or alone (C and F) and run on a BioGel A100 5-ml gel filtration column to resolve protein bound to DNA (fractions 10 to 15) from free protein (fractions 16 to 30). 35 S-NS5BΔ21 stably interacted with ssDNA coated with RPA but not with ssDNA alone or ssDNA coated with T4 gp32. 35 S-NS3h, on the other hand, did not show a stable interaction with ssDNA alone or when coated with RPA or T4 gp32.

    Journal: Journal of Virology

    Article Title: Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity In Vitro

    doi: 10.1128/JVI.01677-14

    Figure Lengend Snippet: RPA ties NS5BΔ21 to the template. 35 S-labeled NS5BΔ21 and NS3h, individually synthesized in transcription/translation reactions in vitro in the presence of [ 35 S]methionine, were incubated with circular ssM13mp18 DNA coated with RPA (A and D) or gp32 (B and E) or alone (C and F) and run on a BioGel A100 5-ml gel filtration column to resolve protein bound to DNA (fractions 10 to 15) from free protein (fractions 16 to 30). 35 S-NS5BΔ21 stably interacted with ssDNA coated with RPA but not with ssDNA alone or ssDNA coated with T4 gp32. 35 S-NS3h, on the other hand, did not show a stable interaction with ssDNA alone or when coated with RPA or T4 gp32.

    Article Snippet: M13mp18 single-stranded DNA (ssDNA) and restriction enzymes were obtained from New England BioLabs (Ipswich, MA).

    Techniques: Recombinase Polymerase Amplification, Labeling, Synthesized, In Vitro, Incubation, Filtration, Stable Transfection

    RPA Levels Differentially Affect Lagging- and Leading-Strand Priming (A) Primase assay. RPA was pre-bound to unprimed (left) and primed (right) M13mp18 ssDNA for 10 min before addition of Pol α for 20 min. 120 nM RPA is saturating assuming a binding footprint of 30 nt. (B and C) Two-dimensional gels of replication assays performed on the 3 kb CPD LEAD template with 10 nM (B) or 100 nM (C) RPA. Lane profiles showing the constituents (denaturing) of the full-length products are shown below each gel. (D) RPA titration on an AhdI-linearized undamaged template. (E) RPA titration on a truncated undamaged template as illustrated.

    Journal: Molecular Cell

    Article Title: The Initial Response of a Eukaryotic Replisome to DNA Damage

    doi: 10.1016/j.molcel.2018.04.022

    Figure Lengend Snippet: RPA Levels Differentially Affect Lagging- and Leading-Strand Priming (A) Primase assay. RPA was pre-bound to unprimed (left) and primed (right) M13mp18 ssDNA for 10 min before addition of Pol α for 20 min. 120 nM RPA is saturating assuming a binding footprint of 30 nt. (B and C) Two-dimensional gels of replication assays performed on the 3 kb CPD LEAD template with 10 nM (B) or 100 nM (C) RPA. Lane profiles showing the constituents (denaturing) of the full-length products are shown below each gel. (D) RPA titration on an AhdI-linearized undamaged template. (E) RPA titration on a truncated undamaged template as illustrated.

    Article Snippet: Primase assay Primed template was prepared by annealing oligonucleotide JY180 (500 nM) (sequence: ) to M13mp18 ssDNA (50 nM) (New England Biolabs) in 10 mM Tris-Cl pH 7.6, 5 mM EDTA and 100 mM NaCl.

    Techniques: Recombinase Polymerase Amplification, Binding Assay, Titration

    a) Illustration of the DNA-enzyme complex captured in a nanopore (left). The base-by-base processive behavior of the ATP-fueled ratcheting enzyme leads to the depicted ionic currents (right) which are discretized to facilitate subsequent analysis (red line). b) Summary analysis of a sequencing run of M13mp18 DNA on an Oxford MinION device demonstrating the available depth of coverage at moderate accuracy. Each data point represents an entire M13 DNA molecule. c) A plot of the mean currents and standard deviations of the 1024 distinct 5-mer sequences, with the full Gaussian distributions of a few example 5-mers shown in blue. d) Depiction of the alignment issues caused by possible detection errors in multiple reads (grey) against the expected ideal current levels (black), including missed levels (red) and extra levels (green).

    Journal: Nature biotechnology

    Article Title: De novo sequencing and variant calling with nanopores using PoreSeq

    doi: 10.1038/nbt.3360

    Figure Lengend Snippet: a) Illustration of the DNA-enzyme complex captured in a nanopore (left). The base-by-base processive behavior of the ATP-fueled ratcheting enzyme leads to the depicted ionic currents (right) which are discretized to facilitate subsequent analysis (red line). b) Summary analysis of a sequencing run of M13mp18 DNA on an Oxford MinION device demonstrating the available depth of coverage at moderate accuracy. Each data point represents an entire M13 DNA molecule. c) A plot of the mean currents and standard deviations of the 1024 distinct 5-mer sequences, with the full Gaussian distributions of a few example 5-mers shown in blue. d) Depiction of the alignment issues caused by possible detection errors in multiple reads (grey) against the expected ideal current levels (black), including missed levels (red) and extra levels (green).

    Article Snippet: M13 Restriction Digest Four micrograms of M13mp18 RFI (New England Biolabs, cat. no. N4018S) DNA were digested with EcoRI restriction enzyme in a 100 microliter reaction volume for 2 hrs at 37C, and then heated for 30 min at 65C to inactivate the enzyme.

    Techniques: Sequencing

    a) Accuracy results of running our code on nanopore data from M13, λ, and E. coli DNA to obtain complete de novo sequences. For M13, error bars indicate the upper and lower bounds for accuracy across 20 random subsets at the given coverage. The green line is the result of error correction and assembly with PBcR using only the 2D basecalled sequences; the red line shows the improvement when we error-correct with the raw data. b) Fraction of single-base variants of M13mp18 correctly called as a function of coverage. Variant sequences were generated by computationally making every possible insertion, deletion, or mutation in the original sequence of M13. A correct call is defined as the original M13 sequences' likelihood being larger than the variant in question. Error bars denote the deviation across 20 random subsets of molecules. c) Variant calling performance of our code on substitution mutations introduced in M13 at a higher frequency of 1%, at a range of coverages. Precision and recall denote the probabilities of false positives and negatives, respectively. The maximum F -score accuracy shown is 99.1% at 16× coverage.

    Journal: Nature biotechnology

    Article Title: De novo sequencing and variant calling with nanopores using PoreSeq

    doi: 10.1038/nbt.3360

    Figure Lengend Snippet: a) Accuracy results of running our code on nanopore data from M13, λ, and E. coli DNA to obtain complete de novo sequences. For M13, error bars indicate the upper and lower bounds for accuracy across 20 random subsets at the given coverage. The green line is the result of error correction and assembly with PBcR using only the 2D basecalled sequences; the red line shows the improvement when we error-correct with the raw data. b) Fraction of single-base variants of M13mp18 correctly called as a function of coverage. Variant sequences were generated by computationally making every possible insertion, deletion, or mutation in the original sequence of M13. A correct call is defined as the original M13 sequences' likelihood being larger than the variant in question. Error bars denote the deviation across 20 random subsets of molecules. c) Variant calling performance of our code on substitution mutations introduced in M13 at a higher frequency of 1%, at a range of coverages. Precision and recall denote the probabilities of false positives and negatives, respectively. The maximum F -score accuracy shown is 99.1% at 16× coverage.

    Article Snippet: M13 Restriction Digest Four micrograms of M13mp18 RFI (New England Biolabs, cat. no. N4018S) DNA were digested with EcoRI restriction enzyme in a 100 microliter reaction volume for 2 hrs at 37C, and then heated for 30 min at 65C to inactivate the enzyme.

    Techniques: Variant Assay, Generated, Mutagenesis, Sequencing

    In vitro DNA repair synthesis and excision activity in extracts from G0/G1- and S-phase-arrested HCT-116 cells. ( a ) DNA repair synthesis on ε A-containing M13mp18 DNA. The cell-free nuclear extracts (0–60 μg per reaction) were incubated

    Journal: Molecular and cellular biochemistry

    Article Title: Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method

    doi: 10.1007/s11010-008-9737-1

    Figure Lengend Snippet: In vitro DNA repair synthesis and excision activity in extracts from G0/G1- and S-phase-arrested HCT-116 cells. ( a ) DNA repair synthesis on ε A-containing M13mp18 DNA. The cell-free nuclear extracts (0–60 μg per reaction) were incubated

    Article Snippet: The M13mp18 single-stranded DNA (2 μg; Bayou Biolabs, LA) was annealed at 80°C for 5 min with 50-fold molar excess of oligonucleotides containing ε A or A (control) at Eco RI or Pst I sites, which had been phosphorylated previously using T4 polynucleotide kinase (New England Biolabs, MA).

    Techniques: In Vitro, Activity Assay, Incubation

    Cell cycle progression of HCT-116 cells after transfection of M13mp18 DNA constructs for in vivo repair at different time points. ( a ) Cells were transfected for repair analysis as described in , then fixed, stained with Propidium Iodide, and examined

    Journal: Molecular and cellular biochemistry

    Article Title: Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method

    doi: 10.1007/s11010-008-9737-1

    Figure Lengend Snippet: Cell cycle progression of HCT-116 cells after transfection of M13mp18 DNA constructs for in vivo repair at different time points. ( a ) Cells were transfected for repair analysis as described in , then fixed, stained with Propidium Iodide, and examined

    Article Snippet: The M13mp18 single-stranded DNA (2 μg; Bayou Biolabs, LA) was annealed at 80°C for 5 min with 50-fold molar excess of oligonucleotides containing ε A or A (control) at Eco RI or Pst I sites, which had been phosphorylated previously using T4 polynucleotide kinase (New England Biolabs, MA).

    Techniques: Transfection, Construct, In Vivo, Staining

    Sensitivity of M13mp18 constructs containing ε A to restriction enzyme digestions. Standard M13mp18 RF-DNA was either undigested (ln 3) or digested with Eco RI (ln 4) and Sac I (ln 5). The CCC M13mp18 carrying no modification (control) at Eco RI

    Journal: Molecular and cellular biochemistry

    Article Title: Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method

    doi: 10.1007/s11010-008-9737-1

    Figure Lengend Snippet: Sensitivity of M13mp18 constructs containing ε A to restriction enzyme digestions. Standard M13mp18 RF-DNA was either undigested (ln 3) or digested with Eco RI (ln 4) and Sac I (ln 5). The CCC M13mp18 carrying no modification (control) at Eco RI

    Article Snippet: The M13mp18 single-stranded DNA (2 μg; Bayou Biolabs, LA) was annealed at 80°C for 5 min with 50-fold molar excess of oligonucleotides containing ε A or A (control) at Eco RI or Pst I sites, which had been phosphorylated previously using T4 polynucleotide kinase (New England Biolabs, MA).

    Techniques: Construct, Countercurrent Chromatography, Modification

    MPG-mediated in vitro excision activity ( a ), immunodetection of MPG ( b ), and in vivo repair kinetics of ε A ( c ) in colon cancer HCT-116 cells. Four micrograms of CCC M13mp18 DNA carrying no modification or ε A at Eco RI site was transfected

    Journal: Molecular and cellular biochemistry

    Article Title: Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method

    doi: 10.1007/s11010-008-9737-1

    Figure Lengend Snippet: MPG-mediated in vitro excision activity ( a ), immunodetection of MPG ( b ), and in vivo repair kinetics of ε A ( c ) in colon cancer HCT-116 cells. Four micrograms of CCC M13mp18 DNA carrying no modification or ε A at Eco RI site was transfected

    Article Snippet: The M13mp18 single-stranded DNA (2 μg; Bayou Biolabs, LA) was annealed at 80°C for 5 min with 50-fold molar excess of oligonucleotides containing ε A or A (control) at Eco RI or Pst I sites, which had been phosphorylated previously using T4 polynucleotide kinase (New England Biolabs, MA).

    Techniques: In Vitro, Activity Assay, Immunodetection, In Vivo, Countercurrent Chromatography, Modification, Transfection

    Abf2p truncations and their effect on DNA binding. ( A ) Schematic representation of the Abf2p deletion mutants. The domains are designated as in the text except for the linker, indicated with an ‘L’. Dashed lines indicate deleted regions. ( B ) Titration of Abf2p and Mut1 to Mut6 constructs binding on linearized M13mp18 dsDNA (8 fmol) at 25°C, by EMSA. The protein/bp ratios are indicated and correspond respectively to 0.37, 0.75, 1.5, 3, 6 and 12 pmol of Abf2p. ( C ) Titration of Abf2p and Mut1 to Mut6 binding to four-way junction DNA (100 nM) by EMSA. ( D ) Effect of Abf2p truncations in vivo . Spot assay of cells with 1/5 serial dilutions on plate containing glycerol as carbon-source (‘YPG plate’) shows the effect of different Abf2p truncations on the ability to maintain y-mtDNA and thus on capability of yeast ( Saccharomyces cerevisiae ) cells to use non-fermentable carbon source. Full-length Abf2p and Mut6 (Abf2p without HMG-box 2) are capable of protecting mtDNA whereas Mut2 (Abf2p without N-flag and N-helix), Mut3 (N-helix+HMG-box 1) and Mut5 (HMG-box 2 alone) are not functional. ( E ) Visualization of mitochondrial nucleoids in cells harboring different Abf2p truncations by DAPI staining of DNA and fluorescence confocal microscopy. Abf2p and Mut6 maintain mtDNA whereas Mut2, Mut3 and Mut5 cannot. C (+) corresponds to wild-type haploids, and C (-) to Abf2 Δcells. The scale bars correspond to 2.5 μm.

    Journal: Nucleic Acids Research

    Article Title: DNA structure directs positioning of the mitochondrial genome packaging protein Abf2p

    doi: 10.1093/nar/gkw1147

    Figure Lengend Snippet: Abf2p truncations and their effect on DNA binding. ( A ) Schematic representation of the Abf2p deletion mutants. The domains are designated as in the text except for the linker, indicated with an ‘L’. Dashed lines indicate deleted regions. ( B ) Titration of Abf2p and Mut1 to Mut6 constructs binding on linearized M13mp18 dsDNA (8 fmol) at 25°C, by EMSA. The protein/bp ratios are indicated and correspond respectively to 0.37, 0.75, 1.5, 3, 6 and 12 pmol of Abf2p. ( C ) Titration of Abf2p and Mut1 to Mut6 binding to four-way junction DNA (100 nM) by EMSA. ( D ) Effect of Abf2p truncations in vivo . Spot assay of cells with 1/5 serial dilutions on plate containing glycerol as carbon-source (‘YPG plate’) shows the effect of different Abf2p truncations on the ability to maintain y-mtDNA and thus on capability of yeast ( Saccharomyces cerevisiae ) cells to use non-fermentable carbon source. Full-length Abf2p and Mut6 (Abf2p without HMG-box 2) are capable of protecting mtDNA whereas Mut2 (Abf2p without N-flag and N-helix), Mut3 (N-helix+HMG-box 1) and Mut5 (HMG-box 2 alone) are not functional. ( E ) Visualization of mitochondrial nucleoids in cells harboring different Abf2p truncations by DAPI staining of DNA and fluorescence confocal microscopy. Abf2p and Mut6 maintain mtDNA whereas Mut2, Mut3 and Mut5 cannot. C (+) corresponds to wild-type haploids, and C (-) to Abf2 Δcells. The scale bars correspond to 2.5 μm.

    Article Snippet: M13mp18 dsDNA plasmids (Bayou Biolabs ) were linearized with BsrB I (New England Biolabs) and purified with the illustra GFX PCR DNA purification kit (GE Healthcare).

    Techniques: Binding Assay, Titration, Construct, In Vivo, Spot Test, Functional Assay, Staining, Fluorescence, Confocal Microscopy

    Chain-formation activity of HLTF with the multiply primed M13mp18 ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.

    Journal: Nucleic Acids Research

    Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

    doi: 10.1093/nar/gky943

    Figure Lengend Snippet: Chain-formation activity of HLTF with the multiply primed M13mp18 ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.

    Article Snippet: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

    Techniques: Activity Assay, Titration, Recombinase Polymerase Amplification

    Suppression of the chain-formation activity of HLTF by interaction with RFC and PCNA. ( A ) Schematic of the experiments. Proteins were sequentially assembled on multiply primed M13mp18 ssDNA tethered to magnetic beads, and ubiquitin ligase assays were performed under standard assay conditions with the protein-bound DNA on the magnetic beads. (B, C) Western blot analysis of the assembled proteins, and ubiquitin chains generated by DNA-bound HLTF ( B ) and his HLTF ΔN ( C ) using anti-RFC1 (upper panels), anti-PCNA (second panels), anti-HLTF (third panels), and anti-ubiquitin antibodies (bottom panels). ‘–’ represents omitted proteins. ‘ΔN’ in RFC represents a mutant RFC consisting of ΔN555 RFC1. ‘FA’ represents the his HLTF FA mutant. ‘ΔN’ in HLTF represents his HLTF ΔN . Each signal intensity (SI) (%) under the HLTF blotting panels in (B) and (C) indicates the relative intensity of HLTF signals after normalization as shown, and that under the ubiquitin blotting panels in (B) and (C) indicates the relative intensity of signals in each plot larger than 60 kDa after normalization as shown. ND, not determined because signal levels were indistinguishable from the background. Relative specific activity (%) was calculated as [SI (%) of ubiquitin blot]/[SI (%) of HLTF blot] × 100. ( D ) Schematic representation of the structures of RFC1 and HLTF and their mutants. ( E ) Alignment of putative APIM sequences of HLTF homologues. The accession numbers of the sequences were NP_001305864 ( H. sapiens ), NP_033236 ( M. musculus ), NP_001179215 ( B. taurus ), XP_005510651 ( C. livia ), XP_018117635 ( X. laevis ), and XP_005163433 ( D. rerio ). ( F ) Effects of HLTF and his HLTF ΔN on singly primed ss M13mp18 DNA replication with pol δ. Reaction mixtures containing RPA, RFC, PCNA, RAD6-RAD18, MMS2-UBC13, and ubiquitin in the presence or absence of E1 and HLTF as indicated, but lacking pol δ, were preincubated at 30°C for 1 min, and DNA synthesis was started by addition of pol δ. Reactions were performed at 30°C for 10 min. The amounts of HLTF and his HLTF ΔN were increased in the order of 0.55, 1.1, and 2.2 pmol. The reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. ‘–’ indicates omitted proteins. ( G ) The radioactivity of [α- 32 P]dCMP incorporated into DNA was measured and normalized to the levels without HLTF.

    Journal: Nucleic Acids Research

    Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

    doi: 10.1093/nar/gky943

    Figure Lengend Snippet: Suppression of the chain-formation activity of HLTF by interaction with RFC and PCNA. ( A ) Schematic of the experiments. Proteins were sequentially assembled on multiply primed M13mp18 ssDNA tethered to magnetic beads, and ubiquitin ligase assays were performed under standard assay conditions with the protein-bound DNA on the magnetic beads. (B, C) Western blot analysis of the assembled proteins, and ubiquitin chains generated by DNA-bound HLTF ( B ) and his HLTF ΔN ( C ) using anti-RFC1 (upper panels), anti-PCNA (second panels), anti-HLTF (third panels), and anti-ubiquitin antibodies (bottom panels). ‘–’ represents omitted proteins. ‘ΔN’ in RFC represents a mutant RFC consisting of ΔN555 RFC1. ‘FA’ represents the his HLTF FA mutant. ‘ΔN’ in HLTF represents his HLTF ΔN . Each signal intensity (SI) (%) under the HLTF blotting panels in (B) and (C) indicates the relative intensity of HLTF signals after normalization as shown, and that under the ubiquitin blotting panels in (B) and (C) indicates the relative intensity of signals in each plot larger than 60 kDa after normalization as shown. ND, not determined because signal levels were indistinguishable from the background. Relative specific activity (%) was calculated as [SI (%) of ubiquitin blot]/[SI (%) of HLTF blot] × 100. ( D ) Schematic representation of the structures of RFC1 and HLTF and their mutants. ( E ) Alignment of putative APIM sequences of HLTF homologues. The accession numbers of the sequences were NP_001305864 ( H. sapiens ), NP_033236 ( M. musculus ), NP_001179215 ( B. taurus ), XP_005510651 ( C. livia ), XP_018117635 ( X. laevis ), and XP_005163433 ( D. rerio ). ( F ) Effects of HLTF and his HLTF ΔN on singly primed ss M13mp18 DNA replication with pol δ. Reaction mixtures containing RPA, RFC, PCNA, RAD6-RAD18, MMS2-UBC13, and ubiquitin in the presence or absence of E1 and HLTF as indicated, but lacking pol δ, were preincubated at 30°C for 1 min, and DNA synthesis was started by addition of pol δ. Reactions were performed at 30°C for 10 min. The amounts of HLTF and his HLTF ΔN were increased in the order of 0.55, 1.1, and 2.2 pmol. The reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. ‘–’ indicates omitted proteins. ( G ) The radioactivity of [α- 32 P]dCMP incorporated into DNA was measured and normalized to the levels without HLTF.

    Article Snippet: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

    Techniques: Activity Assay, Magnetic Beads, Western Blot, Generated, Mutagenesis, Recombinase Polymerase Amplification, DNA Synthesis, Agarose Gel Electrophoresis, Radioactivity

    Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).

    Journal: Nucleic Acids Research

    Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

    doi: 10.1093/nar/gky943

    Figure Lengend Snippet: Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).

    Article Snippet: M13mp18 single-stranded (ss)DNA, M13mp18 RF I, and lambda DNA were purchased from Takara Bio Inc. Multiple-primed M13mp18 ssDNA was generated by annealing 20 synthetic 36-mer oligonucleotides , and an amount corresponding to 150 pmol nucleotides of M13mp18 ssDNA backbone (0.02 pmol) containing 0.41 pmol of the 3′-OH was used in each assay unless otherwise indicated.

    Techniques: Modification, DNA Binding Assay, Incubation, Magnetic Beads, Western Blot, Binding Assay