s1 nuclease Thermo Fisher Search Results


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    Thermo Fisher s1 digestion
    Enzymatic probing of the wild-type (WT) and mutant A3243G hs mt tRNA Leu(UUR) . ( A ) Normalized histograms generated from PAGE analysis of enzymatic cleavage of wild-type (dashed) and mutant A3243G (solid) hs mt tRNA Leu(UUR) using <t>nuclease</t> S1 under nondenaturing
    S1 Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher s1 nuclease buffer
    Detection of ssDNA gaps on ongoing replication forks. ( A ) Neutral comet assay with or without ssDNA-specific S1 endonuclease 24 h after 0 or 5 J/m 2 in XP-C cells depleted for TLS Pols with shRNA. Results are expressed as tail moment (average ± SEM) from ≥ 50 comets per condition of four independent experiments performed in duplicate. The significance of differences between UV-exposed cells compared to its respective untreated control, irradiated cells with or without S1, and between cell lines was assessed by one-way ANOVA followed by Bonferroni test. ( B ) Scheme of DNA fiber assay with the ssDNA-specific nuclease S1 for the detection of ssDNA gaps on ongoing forks. DNA fiber assay with or without <t>S1</t> nuclease in XP-C cor ( C ), XP-C shCT and XP-C shRev3L cells ( D ) and XP-C shCT and XP-C shRev1 ( E ) upon 0 or 20 J/m 2 UVC and represented by CldU/IdU ratios (average ± SEM) from two independent experiments each (≥100 fibers for 0 J/m 2 and ≥150 fibers for 20 J/m 2 ). Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P
    S1 Nuclease Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/s1 nuclease buffer/product/Thermo Fisher
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    Image Search Results


    Enzymatic probing of the wild-type (WT) and mutant A3243G hs mt tRNA Leu(UUR) . ( A ) Normalized histograms generated from PAGE analysis of enzymatic cleavage of wild-type (dashed) and mutant A3243G (solid) hs mt tRNA Leu(UUR) using nuclease S1 under nondenaturing

    Journal: RNA

    Article Title: Structural probing of a pathogenic tRNA dimer

    doi: 10.1261/rna.7143305

    Figure Lengend Snippet: Enzymatic probing of the wild-type (WT) and mutant A3243G hs mt tRNA Leu(UUR) . ( A ) Normalized histograms generated from PAGE analysis of enzymatic cleavage of wild-type (dashed) and mutant A3243G (solid) hs mt tRNA Leu(UUR) using nuclease S1 under nondenaturing

    Article Snippet: Probing experiments were performed on both the wild-type and mutant A3243G hs mt tRNALeu(UUR) transcripts using 25 and 6 × 10−2 U of nuclease S1 (Fermentas) and RNase T2 (Sigma), respectively.

    Techniques: Mutagenesis, Generated, Polyacrylamide Gel Electrophoresis

    CRISPR interference leads to appearance of S1 nuclease sensitive target DNA in vivo . ( A ) Three E. coli strains used are shown. KD263 is capable of both CRISPR interference and adaptation. KD454 is incapable of CRISPR interference. BW40297 is capable of interference but not adaptation. ( B ) Total DNA was prepared from cells shown in A transformed with pG8mut and grown in the presence or in the absence of cas gene expression inducers. Half of each sample was treated with S1 nuclease. qPCR with primer pairs amplifying a 210 nucleotide plasmid fragment or 138 nucleotide CRISPR leader fragment was performed. For each sample, Ct values are presented. For KD263 and BW40297 samples, carrying pG8mut plasmid, higher Ct values observed for plasmid amplicon in the presence of inducers are due to CRISPR interference.

    Journal: Nucleic Acids Research

    Article Title: Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation

    doi: 10.1093/nar/gkx097

    Figure Lengend Snippet: CRISPR interference leads to appearance of S1 nuclease sensitive target DNA in vivo . ( A ) Three E. coli strains used are shown. KD263 is capable of both CRISPR interference and adaptation. KD454 is incapable of CRISPR interference. BW40297 is capable of interference but not adaptation. ( B ) Total DNA was prepared from cells shown in A transformed with pG8mut and grown in the presence or in the absence of cas gene expression inducers. Half of each sample was treated with S1 nuclease. qPCR with primer pairs amplifying a 210 nucleotide plasmid fragment or 138 nucleotide CRISPR leader fragment was performed. For each sample, Ct values are presented. For KD263 and BW40297 samples, carrying pG8mut plasmid, higher Ct values observed for plasmid amplicon in the presence of inducers are due to CRISPR interference.

    Article Snippet: After treating total DNA prepared from induced an uninduced cultures with S1 nuclease, qPCR was performed with a primer pair amplifying a 210 nt pG8mut fragment containing the HS1 protospacer.

    Techniques: CRISPR, In Vivo, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification

    PFGE analysis of bla NDM-1 -positive strains digested with S1 nuclease and hybridization with the pcoA gene probe (a) and silC gene probe (b). (a) Isolate order of lanes 1 to 14: N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, and N14. (b) Isolate order of lanes 1 to 14: N16, N17, N18, N19, N20, N21, N22, N23, 3, 26, N27, N28, N29, N31.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Heavy Metal Resistance Genes Are Associated with blaNDM-1- and blaCTX-M-15-Carrying Enterobacteriaceae

    doi: 10.1128/AAC.02642-17

    Figure Lengend Snippet: PFGE analysis of bla NDM-1 -positive strains digested with S1 nuclease and hybridization with the pcoA gene probe (a) and silC gene probe (b). (a) Isolate order of lanes 1 to 14: N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, and N14. (b) Isolate order of lanes 1 to 14: N16, N17, N18, N19, N20, N21, N22, N23, 3, 26, N27, N28, N29, N31.

    Article Snippet: In this study, the locations of the pcoA , merA , silC , and arsA genes were analyzed by pulsed-field gel electrophoresis with S1 nuclease (S1-PFGE) (Invitrogen Abingdon, UK).

    Techniques: Hybridization

    S1 nuclease protection analysis of the KSHV latency promoter. Shown is an autoradiogram of a 12% denaturing acrylamide gel. Lane 1 shows the input, undigested probe; lanes 2 to 4 show the protected fragments resulting from hybridization to 30 μg of BCBL RNA (lane 2), 15 μg of BCBL-1 RNA (lane 3), or 30 μg of yeast RNA (lane 4) followed by S1 nuclease digestion at 28°C. The arrows point to the two most prominent protected fragments, and size standards are indicated on the left. The asterisks denote the site of the radiolabel.

    Journal: Journal of Virology

    Article Title: A Cluster of Latently Expressed Genes in Kaposi's Sarcoma-Associated Herpesvirus

    doi:

    Figure Lengend Snippet: S1 nuclease protection analysis of the KSHV latency promoter. Shown is an autoradiogram of a 12% denaturing acrylamide gel. Lane 1 shows the input, undigested probe; lanes 2 to 4 show the protected fragments resulting from hybridization to 30 μg of BCBL RNA (lane 2), 15 μg of BCBL-1 RNA (lane 3), or 30 μg of yeast RNA (lane 4) followed by S1 nuclease digestion at 28°C. The arrows point to the two most prominent protected fragments, and size standards are indicated on the left. The asterisks denote the site of the radiolabel.

    Article Snippet: Hybridization (12 h) and S1 nuclease digestion (1 h) were performed at 28°C on 30 μg of total BCBL-1 RNA or yeast RNA by using 3′-32 P-labeled oligonucleotide 7325 (5′-GACGTGACTGCTTCGTGGCGCAGCTGCCTCCAAATGATACACACAT; nt 127851 to 127896) and the S1-Kit (Ambion, Inc., Austin, Tex.) according to the manufacturer’s procedures.

    Techniques: Acrylamide Gel Assay, Hybridization

    S1 nuclease protection assay to map the 5′ end of the K12 transcript. (A) Schematic diagram of the S1 nuclease protection assay. The single-stranded [ 32 ) nt 123983 to 123303 and extends into the K12 intron (nt 123593 to 118800). After S1 nuclease digestion, a ∼250-bp fragment is protected. nt 123842 is the initiation site of the K12 transcript as determined by S1 nuclease protection. The asterisk indicates the 32 P radiolabel. (B) S1 nuclease protection assay. Radiolabeled probe was incubated with PEL tumor poly(A) RNA (lane 1), yeast RNA (lanes 2 and 3), or alone (lane 4) and digested with S1 nuclease (lanes 1 and 2). The protected ∼250-nt fragment in lane 1 is indicated by the arrow.

    Journal: Journal of Virology

    Article Title: The Kaposi's Sarcoma-Associated Herpesvirus K12 Transcript from a Primary Effusion Lymphoma Contains Complex Repeat Elements, Is Spliced, and Initiates from a Novel Promoter

    doi: 10.1128/JVI.76.23.11880-11888.2002

    Figure Lengend Snippet: S1 nuclease protection assay to map the 5′ end of the K12 transcript. (A) Schematic diagram of the S1 nuclease protection assay. The single-stranded [ 32 ) nt 123983 to 123303 and extends into the K12 intron (nt 123593 to 118800). After S1 nuclease digestion, a ∼250-bp fragment is protected. nt 123842 is the initiation site of the K12 transcript as determined by S1 nuclease protection. The asterisk indicates the 32 P radiolabel. (B) S1 nuclease protection assay. Radiolabeled probe was incubated with PEL tumor poly(A) RNA (lane 1), yeast RNA (lanes 2 and 3), or alone (lane 4) and digested with S1 nuclease (lanes 1 and 2). The protected ∼250-nt fragment in lane 1 is indicated by the arrow.

    Article Snippet: S1 nuclease protection was performed with the S1 Assay kit (Ambion) with 5 μg of PEL poly(A) RNA.

    Techniques: Incubation

    Summary of nuclease sensitivity of the heat-treated Rif1BS-derived duplex DNAs. Portions of the sequences of the Rif1BS I:2663 and Rif1BS II:4255 are shown. Locations of S1 nuclease sensitivities and those of protection from DNase I digestion are shown for both strands, as indicated in the key .

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences

    doi: 10.1074/jbc.RA118.005240

    Figure Lengend Snippet: Summary of nuclease sensitivity of the heat-treated Rif1BS-derived duplex DNAs. Portions of the sequences of the Rif1BS I:2663 and Rif1BS II:4255 are shown. Locations of S1 nuclease sensitivities and those of protection from DNase I digestion are shown for both strands, as indicated in the key .

    Article Snippet: For DNase I digestion, 32 P-end–labeled DNA (heat-treated in 50 m m KCl and 40% PEG 200 or nontreated) was digested with DNase I (Takara; 5 units/μl; 1 μl of 1:1000 to 1:5000 dilution (0.005–0.001 units) was used per assay) in 10 μl of 1× DNA binding buffer containing 8 m m MgOAc and 0.5 m m CaCl2 at room temperature for 2.5 min. For S1 nuclease mapping, DNA was digested with S1 nuclease (Thermo Fisher Scientific; 1–3 units) in 10 μl of 10× S1 buffer (supplied with the enzyme) at room temperature for 2.5–5 min. For T7 endonuclease mapping, DNA was digested in 50 m m NaCl, 10 m m Tris-HCl (pH 7.9), 10 m m MgCl2 , and 1 m m DTT with T7 endonuclease I (New England Biolabs; 0.5–5 units) for 10 min at 37 °C in 50 μl.

    Techniques: Derivative Assay

    Ssn6–Tup1-mediated repression is abolished in rpd3 hos1 hos2 cells. ( A ) Endogenous MFA2 RNA levels in the indicated wild-type (WT) or mutant a and α strains were assayed by S1 nuclease protection. A representative gel and averages of MFA2 RNA levels normalized to ACT1 RNA levels from three independent experiments are shown. Fold derepression values reflect the normalized MFA2 signals relative to those observed in wild-type α cells. ( B ) Endogenous SUC2 mRNA levels were assayed by S1 nuclease protection and normalized to ACT1 RNA levels as in ( A ). Fold derepression values reflect the amount of SUC2 signal relative to that observed in wild-type cells under fully repressing conditions (lane 2 ). Values shown are averaged from three independent experiments.

    Journal: Genes & Development

    Article Title: Ssn6-Tup1 interacts with class I histone deacetylases required for repression

    doi:

    Figure Lengend Snippet: Ssn6–Tup1-mediated repression is abolished in rpd3 hos1 hos2 cells. ( A ) Endogenous MFA2 RNA levels in the indicated wild-type (WT) or mutant a and α strains were assayed by S1 nuclease protection. A representative gel and averages of MFA2 RNA levels normalized to ACT1 RNA levels from three independent experiments are shown. Fold derepression values reflect the normalized MFA2 signals relative to those observed in wild-type α cells. ( B ) Endogenous SUC2 mRNA levels were assayed by S1 nuclease protection and normalized to ACT1 RNA levels as in ( A ). Fold derepression values reflect the amount of SUC2 signal relative to that observed in wild-type cells under fully repressing conditions (lane 2 ). Values shown are averaged from three independent experiments.

    Article Snippet: Hybridizations and S1 nuclease digestions were performed as in using 50 U of nuclease (GIBCO BRL).

    Techniques: Mutagenesis

    S1 nuclease mapping of the 5′ endpoints of ery cluster transcripts. The nucleotide(s) at the side of each panel indicate the likely transcription start site(s). (A) eryBIV ; (B) eryAI ; (C) eryBI ; (D) eryBIII ; (E) eryBVI ; (F) eryK . The same primer was used to generate the sequence ladder and the 32 P-labeled probe for S1 assays. For clarity, similar-intensity images of the S1 and sequence ladder lanes from the same gel were juxtaposed.

    Journal: Journal of Bacteriology

    Article Title: Transcriptional Organization of the Erythromycin Biosynthetic Gene Cluster of Saccharopolyspora erythraea

    doi:

    Figure Lengend Snippet: S1 nuclease mapping of the 5′ endpoints of ery cluster transcripts. The nucleotide(s) at the side of each panel indicate the likely transcription start site(s). (A) eryBIV ; (B) eryAI ; (C) eryBI ; (D) eryBIII ; (E) eryBVI ; (F) eryK . The same primer was used to generate the sequence ladder and the 32 P-labeled probe for S1 assays. For clarity, similar-intensity images of the S1 and sequence ladder lanes from the same gel were juxtaposed.

    Article Snippet: Single-stranded DNA probes for S1 nuclease protection assays were generated by a modification of the runoff replication procedure described in the manual accompanying the S1 nuclease kit (Ambion).

    Techniques: Sequencing, Labeling

    Comparison by S1 nuclease protection assay of the mRNA levels of the eryAI transcript in S. erythraea NRRL2338, NRRL2338 eryAI::t rrn , CA340, and CA340 eryAI::t rrn . Lanes: (1) probe only, untreated; (2) probe only, S1 treated; (3) probe hybridized with 40 μg of Saccharomyces cerevisiae RNA; (4) probe hybridized with 40 μg of S. erythraea CA340 RNA; (5) probe hybridized with 40 μg of S. erythraea CA340 eryAI::t rrn RNA; (6) probe hybridized with 40 μg of S. erythraea NRRL2338 eryAI::t rrn RNA; (7) probe hybridized with 40 μg of NRRL2338 RNA. A total of 10 4 cpm of probe was used per S1 nuclease reaction. The asterisk indicates full-length probe. The arrow at the right indicates the S1-protected fragment. For clarity, a lower-intensity exposure of lane 1 was used.

    Journal: Journal of Bacteriology

    Article Title: Transcriptional Organization of the Erythromycin Biosynthetic Gene Cluster of Saccharopolyspora erythraea

    doi:

    Figure Lengend Snippet: Comparison by S1 nuclease protection assay of the mRNA levels of the eryAI transcript in S. erythraea NRRL2338, NRRL2338 eryAI::t rrn , CA340, and CA340 eryAI::t rrn . Lanes: (1) probe only, untreated; (2) probe only, S1 treated; (3) probe hybridized with 40 μg of Saccharomyces cerevisiae RNA; (4) probe hybridized with 40 μg of S. erythraea CA340 RNA; (5) probe hybridized with 40 μg of S. erythraea CA340 eryAI::t rrn RNA; (6) probe hybridized with 40 μg of S. erythraea NRRL2338 eryAI::t rrn RNA; (7) probe hybridized with 40 μg of NRRL2338 RNA. A total of 10 4 cpm of probe was used per S1 nuclease reaction. The asterisk indicates full-length probe. The arrow at the right indicates the S1-protected fragment. For clarity, a lower-intensity exposure of lane 1 was used.

    Article Snippet: Single-stranded DNA probes for S1 nuclease protection assays were generated by a modification of the runoff replication procedure described in the manual accompanying the S1 nuclease kit (Ambion).

    Techniques:

    The right flank of the  ery  gene cluster contains two overlapping transcripts from  eryBIV  to  eryBVII . Lane 1, Full-length  eryBVI  probe treated with S1 nuclease; lane 2, same as lane 1 but not treated with S1 nuclease; lane 3, 40 μg of yeast RNA hybridized with probe; lane 4, 40 μg of  S. erythraea  CA340 RNA hybridized with probe; lane 5, 40 μg of  eryBIV::t rrn  RNA hybridized with probe; lane 6, 40 μg of CA340  eryAIII::t rrn  RNA hybridized with probe. All samples were treated with 50 U of S1 nuclease. A total of 3 × 10 4  Chelenkov counts per min were used per reaction. P, full-length protected fragment; the asterisk marks a shortened S1-protected fragment.

    Journal: Journal of Bacteriology

    Article Title: Transcriptional Organization of the Erythromycin Biosynthetic Gene Cluster of Saccharopolyspora erythraea

    doi:

    Figure Lengend Snippet: The right flank of the ery gene cluster contains two overlapping transcripts from eryBIV to eryBVII . Lane 1, Full-length eryBVI probe treated with S1 nuclease; lane 2, same as lane 1 but not treated with S1 nuclease; lane 3, 40 μg of yeast RNA hybridized with probe; lane 4, 40 μg of S. erythraea CA340 RNA hybridized with probe; lane 5, 40 μg of eryBIV::t rrn RNA hybridized with probe; lane 6, 40 μg of CA340 eryAIII::t rrn RNA hybridized with probe. All samples were treated with 50 U of S1 nuclease. A total of 3 × 10 4 Chelenkov counts per min were used per reaction. P, full-length protected fragment; the asterisk marks a shortened S1-protected fragment.

    Article Snippet: Single-stranded DNA probes for S1 nuclease protection assays were generated by a modification of the runoff replication procedure described in the manual accompanying the S1 nuclease kit (Ambion).

    Techniques:

    Partial nucleotide sequences and deduced amino acid sequences of cebREFG and bglC . The deduced amino acid sequences are given in the one-letter code below the nucleotide (nt) sequence, and nucleotide numbers are shown on the right. The putative ribosome binding sites (rbs) are in white letters on a black background, and predicted terminators are indicated by arrows above the sequence. The transcriptional start sites are in boldface type and are marked by asterisks followed by arrows indicating the direction of transcription (t E , transcription start site for cebE ; t R1 and t R2 , transcription start sites for cebR ). The sequence similar to that of the Streptomyces class E promoter is marked by a line above the sequence. The oligonucleotides R103 and E101 used for S1 nuclease mapping are represented by broken lines. The predicted HTH motif in the deduced CebR protein is underscored, and the putative operator sequence for CebR binding is boxed (O CebR ). The signal peptide of CebE followed by the recognition sequence for the cleavage site of lipoprotein signal peptidase is double underscored. The NH 2 -terminal amino acids of peptides from the purified CebE and BglC proteins determined by Edman degradation are in boldface type. Most of the sequence within the structural genes has been omitted, as is indicated by dots and double-slashed bars.

    Journal: Applied and Environmental Microbiology

    Article Title: Characterization of the Binding Protein-Dependent Cellobiose and Cellotriose Transport System of the Cellulose Degrader Streptomyces reticuli

    doi:

    Figure Lengend Snippet: Partial nucleotide sequences and deduced amino acid sequences of cebREFG and bglC . The deduced amino acid sequences are given in the one-letter code below the nucleotide (nt) sequence, and nucleotide numbers are shown on the right. The putative ribosome binding sites (rbs) are in white letters on a black background, and predicted terminators are indicated by arrows above the sequence. The transcriptional start sites are in boldface type and are marked by asterisks followed by arrows indicating the direction of transcription (t E , transcription start site for cebE ; t R1 and t R2 , transcription start sites for cebR ). The sequence similar to that of the Streptomyces class E promoter is marked by a line above the sequence. The oligonucleotides R103 and E101 used for S1 nuclease mapping are represented by broken lines. The predicted HTH motif in the deduced CebR protein is underscored, and the putative operator sequence for CebR binding is boxed (O CebR ). The signal peptide of CebE followed by the recognition sequence for the cleavage site of lipoprotein signal peptidase is double underscored. The NH 2 -terminal amino acids of peptides from the purified CebE and BglC proteins determined by Edman degradation are in boldface type. Most of the sequence within the structural genes has been omitted, as is indicated by dots and double-slashed bars.

    Article Snippet: For every S1 nuclease protection reaction, 50 μg of RNA was hybridized to ∼105 Cerenkov counts of the PCR fragment in Na-TCA buffer ( ) min−1 at 45°C for 6 h, after denaturation at 65°C for 15 min. Hybridization products were digested with S1 nuclease (Gibco, Life Technologies, Karlsruhe, Germany) as outlined by the manufacturer.

    Techniques: Sequencing, Binding Assay, Purification

    Mapping of the transcription initiation sites of cebE and cebR . RNA (50 μg) prepared from mycelia of S. reticuli grown in the presence of cellobiose was hybridized to 0.1 pmol of the 32 P-labelled cebR (A) or cebE (B) probes, and S1 nuclease treatment (lanes S) was done as described in Materials and Methods. ACGT indicates the cebR and cebE nucleotide sequence ladders. The asterisks mark the most probable transcription start sites.

    Journal: Applied and Environmental Microbiology

    Article Title: Characterization of the Binding Protein-Dependent Cellobiose and Cellotriose Transport System of the Cellulose Degrader Streptomyces reticuli

    doi:

    Figure Lengend Snippet: Mapping of the transcription initiation sites of cebE and cebR . RNA (50 μg) prepared from mycelia of S. reticuli grown in the presence of cellobiose was hybridized to 0.1 pmol of the 32 P-labelled cebR (A) or cebE (B) probes, and S1 nuclease treatment (lanes S) was done as described in Materials and Methods. ACGT indicates the cebR and cebE nucleotide sequence ladders. The asterisks mark the most probable transcription start sites.

    Article Snippet: For every S1 nuclease protection reaction, 50 μg of RNA was hybridized to ∼105 Cerenkov counts of the PCR fragment in Na-TCA buffer ( ) min−1 at 45°C for 6 h, after denaturation at 65°C for 15 min. Hybridization products were digested with S1 nuclease (Gibco, Life Technologies, Karlsruhe, Germany) as outlined by the manufacturer.

    Techniques: Sequencing

    Detection of ssDNA gaps on ongoing replication forks. ( A ) Neutral comet assay with or without ssDNA-specific S1 endonuclease 24 h after 0 or 5 J/m 2 in XP-C cells depleted for TLS Pols with shRNA. Results are expressed as tail moment (average ± SEM) from ≥ 50 comets per condition of four independent experiments performed in duplicate. The significance of differences between UV-exposed cells compared to its respective untreated control, irradiated cells with or without S1, and between cell lines was assessed by one-way ANOVA followed by Bonferroni test. ( B ) Scheme of DNA fiber assay with the ssDNA-specific nuclease S1 for the detection of ssDNA gaps on ongoing forks. DNA fiber assay with or without S1 nuclease in XP-C cor ( C ), XP-C shCT and XP-C shRev3L cells ( D ) and XP-C shCT and XP-C shRev1 ( E ) upon 0 or 20 J/m 2 UVC and represented by CldU/IdU ratios (average ± SEM) from two independent experiments each (≥100 fibers for 0 J/m 2 and ≥150 fibers for 20 J/m 2 ). Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Journal: Nucleic Acids Research

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells

    doi: 10.1093/nar/gkw280

    Figure Lengend Snippet: Detection of ssDNA gaps on ongoing replication forks. ( A ) Neutral comet assay with or without ssDNA-specific S1 endonuclease 24 h after 0 or 5 J/m 2 in XP-C cells depleted for TLS Pols with shRNA. Results are expressed as tail moment (average ± SEM) from ≥ 50 comets per condition of four independent experiments performed in duplicate. The significance of differences between UV-exposed cells compared to its respective untreated control, irradiated cells with or without S1, and between cell lines was assessed by one-way ANOVA followed by Bonferroni test. ( B ) Scheme of DNA fiber assay with the ssDNA-specific nuclease S1 for the detection of ssDNA gaps on ongoing forks. DNA fiber assay with or without S1 nuclease in XP-C cor ( C ), XP-C shCT and XP-C shRev3L cells ( D ) and XP-C shCT and XP-C shRev1 ( E ) upon 0 or 20 J/m 2 UVC and represented by CldU/IdU ratios (average ± SEM) from two independent experiments each (≥100 fibers for 0 J/m 2 and ≥150 fibers for 20 J/m 2 ). Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Article Snippet: For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC.

    Techniques: Neutral Comet Assay, shRNA, Irradiation

    Effects of 6-4PP and CPD photorepair on ssDNA gaps formation and postreplication repair (PRR) tracts. XP-C shCT cells were transduced with Ad6-4phr, AdCPDphr or mock-treated. ( A ) Scheme of DNA fiber assay with the ssDNA-specific S1 nuclease in photorepair conditions. ( B ) CldU/IdU ratios from XP-C cells exposed to 0 or 50 J/m 2 and treated or not with S1 from three independent experiments (≥100 fibers for 0 J/m 2 and ≥150 fibers for 50 J/m 2 each). ( C ) Scheme for PRR tract detection in photorepair conditions. ( D ) Quantification of PRR tracts density 24 h upon exposure to 0 or 20 J/m 2 . Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Journal: Nucleic Acids Research

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells

    doi: 10.1093/nar/gkw280

    Figure Lengend Snippet: Effects of 6-4PP and CPD photorepair on ssDNA gaps formation and postreplication repair (PRR) tracts. XP-C shCT cells were transduced with Ad6-4phr, AdCPDphr or mock-treated. ( A ) Scheme of DNA fiber assay with the ssDNA-specific S1 nuclease in photorepair conditions. ( B ) CldU/IdU ratios from XP-C cells exposed to 0 or 50 J/m 2 and treated or not with S1 from three independent experiments (≥100 fibers for 0 J/m 2 and ≥150 fibers for 50 J/m 2 each). ( C ) Scheme for PRR tract detection in photorepair conditions. ( D ) Quantification of PRR tracts density 24 h upon exposure to 0 or 20 J/m 2 . Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Article Snippet: For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC.

    Techniques: Transduction