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Replication-Independent Repair of DNA Interstrand Crosslinks
Molecular Cell, Volume 47
Supplemental Information Replication-Independent Repair of DNA Interstrand Crosslinks Hannah L. Williams, Max E. Gottesman, and Jean Gautier
Inventory of Supplemental Information Figure S1 – Supplemental figure and legend related to main Figure 1. Figure S1 – Supplemental figure and legend related to main Figure 2. Figure S3 – Supplemental figure and legend related to main Figure3. Supplemental Experimental Procedures – Additional information regarding experimental procedures.
Figure S1.
Figure S1. (A) DNA replication assessed by α32P-dCTP incorporation into DNA showing that plasmid DNA is replicated in HSS + NPE extracts, but not in HSS or NPE alone. (B) Quantification of the X:C ratio of ICL plasmid incubated in HSS. ICL plasmid was digested with AluI following incubation in extract to assess repair on both strands of the DNA. (C) ICL or control plasmids were incubated in HSS in the presence or absence of ATR inhibitor (ATRi). Western blot analysis was performed on soluble extracts with the indicated antibodies. (D) Western blot analysis was performed on mock- and FANCIdepleted HSS with the indicated antibodies to demonstrate efficient depletion of FANCI. (E) Western blot analysis was performed on mock- and Rad51-depleted HSS with the indicated antibodies to demonstrate efficient depletion of Rad51. (F) Western blot analysis was performed on mock- and Rev7-depleted HSS with the indicated antibodies to demonstrate efficient depletion of Rev7.
Figure S2.
Figure S2. (A) Western blot analysis was performed on mock- and Pol -depleted HSS with the indicated antibodies to demonstrate efficient depletion of Pol . (B) Quantification of ICL repair in mock- and Pol -depleted HSS+NPE in the presence or absence of roscovitine. (C) To demonstrate that equal amounts of Pol WT and Pol D199A, E200A
were used in rescue experiments, 0.4 μl of each partially purified in vitro
transcription and translation reaction and a control reaction where no DNA was added (ND) were analyzed by western blotting with Pol antibody. (D) To demonstrate that equal amounts of Pol WT and Pol F562A,F563A were used in rescue experiments, 0.4 μl of each partially purified in vitro transcription and translation reaction and a control reaction where no DNA was added (ND) were analyzed by western blotting with Pol antibody. (E) In vitro pull-down assay. GST tagged Rev1 C-terminus (aa 815-1230) was immobilized on glutathione beads and used to pull down partially purified in vitro transcribed and translated Pol WT and Pol F562A,F563A. Bound proteins were analyzed by western blotting. (F) Purified ICL and control plasmids showing a single, circular DNA form.
Figure S3.
Figure S3. (A) Western blot analysis of mock- and Rad1-depleted HSS with the indicated antibodies to demonstrate efficient depletion of Rad1. (B) Western blot analysis of mock- and PCNA-depleted HSS with the indicated antibodies to demonstrate efficient depletion of PCNA. (C) To demonstrate that equal amounts of Pol WT, Pol D634A,D789A and Pol I857A,F860A,F861A were used in rescue experiments, 0.4 μl of each partially purified in vitro transcription and translation reaction and a control reaction where no DNA was added (ND) were analyzed by western blotting with Pol antibody.
Supplemental Experimental Procedures Pol mutagenesis primers Pol D199A, E200A was made using the primers 5CCTATGAGTCTTGCTGCAGCTTACCTGGACTTC-3 and 5GAAGTCCAGGTAAGCTGCAGCAAGACTCATAGG-3’. The Rev1 interaction mutant Pol F562A, F563A was made using the primers 5-CCT CAG AGA CAG AGC GCT GCT AAT CAG AAG CGA GCA GCC-3 and 5-GGC TGC TCG CTT CTG ATT AGC AGC GCT CTG TCT CTG AGG-3. The UBZ domain mutant Pol D634A,D789A was made using the primers 5-GCAACCTTTAATAAGCACATCGCCAAATGTTTAAGTGGATCACC 3, 5 GGTGATCCACTTAAACATTTGGCGATGTGCTTATTAAAGGTTGC 3, 5 CTTACAGCATTTAACAGACACGTAGCTGTGTGTCTTAATAAAGG 3 and 5 CCTTTATTAAGACACACAGCTACGTGTCTGTTAAATGCTGTAAG 5. The PIP box mutant was made using the primers 5GCCAAATTCTTCCAAAAACACCGCGGACAGAGCCGCCAAGTAGAATTTCAAT GAC-3 and 5GTCATTGAAATTCTACTTGGCGGCTCTGTCCGCGGTGTTTTTGGAAGAATTTG GC-3. In all cases mutant bases are underlined. All mutations were subsequently confirmed by DNA sequencing.
Rev1 cloning and purification The C-terminus of Rev1 was amplified by PCR from the Xenopus clone pCMVSport.ccdb-Rev1 (Open Biosystems accession number BC070743) using the primers 5-
CATCAGCTTGGATCCGTAGGTGGACTG -3 and 5 ‐ GTTAAACAACCCGGGAACAACTTTC -3. The underlined sequences are BamH1 and SmaI restriction sites respectively. The resulting fragment was digested with SmaI and BamHI and cloned into pGex2T using the same enzymes. GST-Rev1 (815-1230) expression was induced with 0.5 mM IPTG for 2hrs at 30 ˚C in BL21 Star™(DE3)pLysS cells. Cells were lysed in 20 mM Tris-Cl pH8, 100 mM NaCl, 1mM EDTA, 1mM DTT, 0.5% NP40, 1mM PMSF and 5μg/μl leupeptin by sonication.
Deletion of the SV40 origin from pEGFP-N3 The SV40 origin of pEGFP-N3 was deleted by performing PCR with the phosphorylated primers 5- GAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAG -3 and 5- GGGCGGAGTTAGGGGCGGGACTATGGTTGCTGAC -3, which hybridize on either side of the SV40 origin such that the resulting linear PCR product does not contain the SV40 origin sequence. The PCR product was ligated to allow re-circularization to yield the modified form of the plasmid pEGFP-N3-ΔSV40.
Antibodies Rad51, FANCI, RPA70 and Pol antibodies were generated by immunizing rabbits with the peptides: CVPMGFTTATEFHQRRSEII, DEENEPDNEQAVTEEESQEPKK, CHKEYSRRLIMNIRKMATQGV and CPASKKSKPNSSKNTIDRFFK respectively. Western blotting analysis for phospho-Chk1, PCNA and H3 was carried out using commercial antibodies from cell signaling (Ser345), Sigma (clone PC10) and Cell signaling (Cat #9715S) respectively. Total Chk1 antibodies were a gift from H. Lindsay,
Rev 7 antibodies were a gift from J. Walter and Rad1 antibodies were a gift from K. Cimprich.
Quantitative PCR qPCR was carried out using the primers 5-TGGCAGCACTGCATAATTCT-3, 5TCTCAACAGCGGTAAGATCC-3 (C primers), 5 AGATAGGGTTGAGTGTTGTTC3 and 5-GCGTCCATTCCATGGTC-3 (X primers). PCR cycling conditions were as follows: 50˚C for 2 min, 95˚C for 15 min and 40 cycles of 95˚C for 15 sec, 62˚C for 30 sec.
Expression of Pol in rabbit reticulocyte lysates 2μg of pCMV-Sport-Pol was incubated in 100 μl of TnT® Sp6 Quick Master Mix with 4 μl 1mM Methionine for 90 min at 30˚C. Reactions were diluted to 400 μl with PBS and polymin-P was added to a concentration of 0.06% and incubated for 30 min at 4˚C with rotation. Precipitated plasmid DNA was removed by centrifugation. 100% Saturated (NH4)2SO4 was added dropwise to the supernatant to a final concentration of 55 % and incubated for 30 min at 4˚C with rotation. Precipitated proteins were recovered by centrifugation for 30 min at 16 000 g and resuspended in 15μl 20 mM HEPES-KOH pH 7.8, 100 mM NaCl and 2% glycerol, and dialyzed for 3 hrs at 4˚C in the same buffer to remove any residual (NH4)2SO4. TnT mastermix lacking DNA, was manipulated using the same method, and added to mock and Δ Pol reactions as a control.
Sequencing repaired ICL plasmid Multiple PCR products spanning a 245 region surrounding the ICL, were amplified from 7 independent ICL repair assays in HSS using HotStar Hifidelity DNA polymerase and the primers 5-AGATAGGGTTGAGTGTTGTTC-3 and 5AGCTATGACCATGATTACGC-3. The resulting amplicon was cloned into pGEM-T Easy (Promega) and a total of 56 white colonies were selected and sequenced using T7 primer.
Supplemental Information Replication-Independent Repair of DNA Interstrand Crosslinks Hannah L. Williams, Max E. Gottesman, and Jean Gautier
Inventory of Supplemental Information Figure S1 – Supplemental figure and legend related to main Figure 1. Figure S1 – Supplemental figure and legend related to main Figure 2. Figure S3 – Supplemental figure and legend related to main Figure3. Supplemental Experimental Procedures – Additional information regarding experimental procedures.
Figure S1.
Figure S1. (A) DNA replication assessed by α32P-dCTP incorporation into DNA showing that plasmid DNA is replicated in HSS + NPE extracts, but not in HSS or NPE alone. (B) Quantification of the X:C ratio of ICL plasmid incubated in HSS. ICL plasmid was digested with AluI following incubation in extract to assess repair on both strands of the DNA. (C) ICL or control plasmids were incubated in HSS in the presence or absence of ATR inhibitor (ATRi). Western blot analysis was performed on soluble extracts with the indicated antibodies. (D) Western blot analysis was performed on mock- and FANCIdepleted HSS with the indicated antibodies to demonstrate efficient depletion of FANCI. (E) Western blot analysis was performed on mock- and Rad51-depleted HSS with the indicated antibodies to demonstrate efficient depletion of Rad51. (F) Western blot analysis was performed on mock- and Rev7-depleted HSS with the indicated antibodies to demonstrate efficient depletion of Rev7.
Figure S2.
Figure S2. (A) Western blot analysis was performed on mock- and Pol -depleted HSS with the indicated antibodies to demonstrate efficient depletion of Pol . (B) Quantification of ICL repair in mock- and Pol -depleted HSS+NPE in the presence or absence of roscovitine. (C) To demonstrate that equal amounts of Pol WT and Pol D199A, E200A
were used in rescue experiments, 0.4 μl of each partially purified in vitro
transcription and translation reaction and a control reaction where no DNA was added (ND) were analyzed by western blotting with Pol antibody. (D) To demonstrate that equal amounts of Pol WT and Pol F562A,F563A were used in rescue experiments, 0.4 μl of each partially purified in vitro transcription and translation reaction and a control reaction where no DNA was added (ND) were analyzed by western blotting with Pol antibody. (E) In vitro pull-down assay. GST tagged Rev1 C-terminus (aa 815-1230) was immobilized on glutathione beads and used to pull down partially purified in vitro transcribed and translated Pol WT and Pol F562A,F563A. Bound proteins were analyzed by western blotting. (F) Purified ICL and control plasmids showing a single, circular DNA form.
Figure S3.
Figure S3. (A) Western blot analysis of mock- and Rad1-depleted HSS with the indicated antibodies to demonstrate efficient depletion of Rad1. (B) Western blot analysis of mock- and PCNA-depleted HSS with the indicated antibodies to demonstrate efficient depletion of PCNA. (C) To demonstrate that equal amounts of Pol WT, Pol D634A,D789A and Pol I857A,F860A,F861A were used in rescue experiments, 0.4 μl of each partially purified in vitro transcription and translation reaction and a control reaction where no DNA was added (ND) were analyzed by western blotting with Pol antibody.
Supplemental Experimental Procedures Pol mutagenesis primers Pol D199A, E200A was made using the primers 5CCTATGAGTCTTGCTGCAGCTTACCTGGACTTC-3 and 5GAAGTCCAGGTAAGCTGCAGCAAGACTCATAGG-3’. The Rev1 interaction mutant Pol F562A, F563A was made using the primers 5-CCT CAG AGA CAG AGC GCT GCT AAT CAG AAG CGA GCA GCC-3 and 5-GGC TGC TCG CTT CTG ATT AGC AGC GCT CTG TCT CTG AGG-3. The UBZ domain mutant Pol D634A,D789A was made using the primers 5-GCAACCTTTAATAAGCACATCGCCAAATGTTTAAGTGGATCACC 3, 5 GGTGATCCACTTAAACATTTGGCGATGTGCTTATTAAAGGTTGC 3, 5 CTTACAGCATTTAACAGACACGTAGCTGTGTGTCTTAATAAAGG 3 and 5 CCTTTATTAAGACACACAGCTACGTGTCTGTTAAATGCTGTAAG 5. The PIP box mutant was made using the primers 5GCCAAATTCTTCCAAAAACACCGCGGACAGAGCCGCCAAGTAGAATTTCAAT GAC-3 and 5GTCATTGAAATTCTACTTGGCGGCTCTGTCCGCGGTGTTTTTGGAAGAATTTG GC-3. In all cases mutant bases are underlined. All mutations were subsequently confirmed by DNA sequencing.
Rev1 cloning and purification The C-terminus of Rev1 was amplified by PCR from the Xenopus clone pCMVSport.ccdb-Rev1 (Open Biosystems accession number BC070743) using the primers 5-
CATCAGCTTGGATCCGTAGGTGGACTG -3 and 5 ‐ GTTAAACAACCCGGGAACAACTTTC -3. The underlined sequences are BamH1 and SmaI restriction sites respectively. The resulting fragment was digested with SmaI and BamHI and cloned into pGex2T using the same enzymes. GST-Rev1 (815-1230) expression was induced with 0.5 mM IPTG for 2hrs at 30 ˚C in BL21 Star™(DE3)pLysS cells. Cells were lysed in 20 mM Tris-Cl pH8, 100 mM NaCl, 1mM EDTA, 1mM DTT, 0.5% NP40, 1mM PMSF and 5μg/μl leupeptin by sonication.
Deletion of the SV40 origin from pEGFP-N3 The SV40 origin of pEGFP-N3 was deleted by performing PCR with the phosphorylated primers 5- GAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAG -3 and 5- GGGCGGAGTTAGGGGCGGGACTATGGTTGCTGAC -3, which hybridize on either side of the SV40 origin such that the resulting linear PCR product does not contain the SV40 origin sequence. The PCR product was ligated to allow re-circularization to yield the modified form of the plasmid pEGFP-N3-ΔSV40.
Antibodies Rad51, FANCI, RPA70 and Pol antibodies were generated by immunizing rabbits with the peptides: CVPMGFTTATEFHQRRSEII, DEENEPDNEQAVTEEESQEPKK, CHKEYSRRLIMNIRKMATQGV and CPASKKSKPNSSKNTIDRFFK respectively. Western blotting analysis for phospho-Chk1, PCNA and H3 was carried out using commercial antibodies from cell signaling (Ser345), Sigma (clone PC10) and Cell signaling (Cat #9715S) respectively. Total Chk1 antibodies were a gift from H. Lindsay,
Rev 7 antibodies were a gift from J. Walter and Rad1 antibodies were a gift from K. Cimprich.
Quantitative PCR qPCR was carried out using the primers 5-TGGCAGCACTGCATAATTCT-3, 5TCTCAACAGCGGTAAGATCC-3 (C primers), 5 AGATAGGGTTGAGTGTTGTTC3 and 5-GCGTCCATTCCATGGTC-3 (X primers). PCR cycling conditions were as follows: 50˚C for 2 min, 95˚C for 15 min and 40 cycles of 95˚C for 15 sec, 62˚C for 30 sec.
Expression of Pol in rabbit reticulocyte lysates 2μg of pCMV-Sport-Pol was incubated in 100 μl of TnT® Sp6 Quick Master Mix with 4 μl 1mM Methionine for 90 min at 30˚C. Reactions were diluted to 400 μl with PBS and polymin-P was added to a concentration of 0.06% and incubated for 30 min at 4˚C with rotation. Precipitated plasmid DNA was removed by centrifugation. 100% Saturated (NH4)2SO4 was added dropwise to the supernatant to a final concentration of 55 % and incubated for 30 min at 4˚C with rotation. Precipitated proteins were recovered by centrifugation for 30 min at 16 000 g and resuspended in 15μl 20 mM HEPES-KOH pH 7.8, 100 mM NaCl and 2% glycerol, and dialyzed for 3 hrs at 4˚C in the same buffer to remove any residual (NH4)2SO4. TnT mastermix lacking DNA, was manipulated using the same method, and added to mock and Δ Pol reactions as a control.
Sequencing repaired ICL plasmid Multiple PCR products spanning a 245 region surrounding the ICL, were amplified from 7 independent ICL repair assays in HSS using HotStar Hifidelity DNA polymerase and the primers 5-AGATAGGGTTGAGTGTTGTTC-3 and 5AGCTATGACCATGATTACGC-3. The resulting amplicon was cloned into pGEM-T Easy (Promega) and a total of 56 white colonies were selected and sequenced using T7 primer.