Kato N , Kawasoe Y , Williams H , Coates E , Roy U , Shi Y , Beese LS , Schärer OD , Yan H , Gottesman ME , Takahashi TS , Gautier J .

P01 CA174653 NCI NIH HHS , R35 CA197606 NCI NIH HHS , P01 CA092584 NCI NIH HHS

	Graphical Abstract
	Figure 1. ICL Sensing by MutSα (MSH2–MSH6) (A) Plasmid pull-down assay of control plasmids or plasmids containing a single site-specific SJG-136 ICL lesion. Plasmids were incubated for 40 min in HSS and then purified from extracts using recombinant Bio-LacR protein coupled to streptavidin beads. Plasmid-bound proteins were analyzed by western blot (WB) using the indicated antibodies. (B) Plasmid pull-down assay and WB of SJG-136-treated plasmids. In the last lane, labeled (10×2), twice the amount of 10 μM-treated plasmids was incubated in HSS. Input was plasmid DNA run on an agarose gel. (C) MSH2–MSH6 immunodepletion. Mock- and MSH2-depleted HSS was analyzed by WB. (D) Quantification of ICL repair in mock- and MSH2-depleted HSS at 3 hr. Results represent mean ± SEM from n = 7 independent experiments. (E) Quantification of ICL repair in HSS and HSS supplemented with MutSαWT at 3 hr. Results represent mean ± SEM from n = 11 independent experiments.
	Figure 2. Mechanism of ICL Recognition by MutSα (MSH2–MSH6) (A) Plasmids containing a single site-specific A:C mismatch were incubated in mock-depleted, MSH2-depleted, or MSH2-depleted HSS supplemented with recombinant MutSαWT or MutSαFXE for the indicated amount of time. Mismatch repair efficiency was measured by digestion with XmnI with BamHI or XhoI restriction enzymes, for A→G repair (top) and C→T repair (bottom), respectively. Data are representative of three independent experiments. (B) Quantification of ICL repair in mock-depleted, MSH2-depleted, or MSH2-depleted HSS supplemented with recombinant MutSαWT (n = 7), MutSαFXE (n = 6), or MutSαPIP (n = 4) at 3 hr. Results represent mean ± SEM of independent experiments. (C) Plasmid pull-down assay and WB of SJG-136-treated plasmids in mock- or PCNA-depleted extract. See also Figure S2.
	Figure 3. Nucleolytic Incision of ICL Lesions by MutLα (MLH1-PMS2) (A) Schematic of nicking sites engineered into ICL pBS plasmids (see also Figure S3A). (B) Quantification of ICL repair in HSS of unnicked, 5′ or 3′ nicked plasmids at 90 min. Results represent mean ± SEM from n = 6 independent experiments. (C) Plasmid pull-down assay and WB of SJG-136-treated plasmids in mock- or MSH2-depleted extract. (D) MLH1-PMS2 immunodepletion. Mock- and MLH1-depleted HSS was analyzed by WB. (E) Quantification of ICL repair in mock-depleted, MLH1-depleted, or MLH1-depleted HSS supplemented with recombinant MutLαWT (n = 12) or MutLαn.d (n = 7) at 90 min. Results represent mean ± SEM independent experiments. (F) Quantification of ICL repair in HSS with overexpression of buffer, MutLαWT, or MutLαn.d. at 90 min. Results represent mean ± SEM from n = 5 independent experiments. See also Figure S3.
	Figure 4. Nucleolytic Processing of ICL Lesions by EXO1 (A) Plasmid pull-down assay and WB of SJG-136-treated plasmids in mock- or MSH2-depleted extract. (B) EXO1 immunodepletion. Mock- and EXO1-depleted HSS was analyzed by WB. (C) Quantification of ICL repair in mock-depleted, EXO1-depleted, or EXO1-depleted HSS supplemented with recombinant EXO1WT or EXO1D173A at 3 hr. Results represent mean ± SEM from n = 7 independent experiments. (D) Exo1352 nuclease assay with control or SJG-treated oligonucleotides. Reaction products were run on a denaturing polyacrylamide gel and stained with SYBR Gold. See also Figure S4.
	Figure 5. ICL Structure and Repair Efficiency (A) Structures of normal B-form DNA (PDB: 1BNA) and DNA duplexes containing trimethylene 5′ GpC-ICL (PDB: 1LUH) and 5′ CpG-ICL (PDB: 2KNK) lesions. (B) Plasmids containing single trimethylene GpC or CpG-ICL were incubated in HSS, and soluble extracts were analyzed by WB. (C) Quantification of ICL repair of trimethylene GpC and CpG plasmids at the indicated time points. Results represent mean ± SEM from n = 4 independent experiments. (D) Quantification of ICL repair of trimethylene GpC and CpG plasmids in mock- and MSH2-depleted HSS at 3 hr. Results represent mean ± SEM from n = 4 independent experiments. (E) Quantification of ICL repair of trimethylene GpC and NM-like ICL plasmid using TaqMan qPCR reagent at the indicated time points. Results represent mean ± SEM from n = 3 independent experiments. See also Figure S5.
	Figure 6. Model for MMR-Mediated ICL Repair ICLs can be recognized and repaired in the absence of replication. The MutSα complex senses and binds ICL lesions and recruits downstream repair proteins including the MutLα endonuclease, PCNA, and EXO1 exonuclease. The ICL lesion is repaired through a multi-step excision resynthesis reaction. The latter requires Polκ and monoubiquitinated PCNA.
	Figure S1. Schematic of ICL plasmids and preparation of SJG-136 treated plasmids, Related to Figure 1 (A) SJG-136 preferentially reacts with DNA at guanine residues at 5’ purine–GATC–pyrimidine sequences to form ICLs. (B) Sequences of trimethylene and SJG-136 ICL oligos that were ligated into pBS to construct plasmids with a single site-specific ICL (left). Schematic of the resulting ICL plasmid. The binding site for Bio-LacR used for plasmid pull-down experiments, and primers used for qPCR to calculate ICL repair are illustrated (right). (C) Schematic representation of pBS treated with SJG-136. The 7 sequences corresponding to SJG-136 ICL forming sites are illustrated. The single SJG-136 reaction site that overlaps with a BamHI recognition sequence 2 is specified. The binding site for Bio-LacR and primers used for quantification of plasmids in plasmid pulldown experiments are indicated. (D) Accumulation of ICLs on SJG-136 treated plasmids assessed using BamHI digestion. Plasmids were run on ethidium-bromide agarose gel before (upper panel) or after BamHI digestion (lower panel). With increasing doses of the drug, plasmids become refractory to BamHI digestion. (E) The number of ICLs induced on plasmids treated with SJG-136 was estimated from quantification of BamHI digestion products. Results represent quantification from 3 independent experiments ± SD. (F) Representative figure of the quantification of plasmids recovered from extracts after pull-down experiments using qPCR. Results represent technical triplicate ± SD from a single representative experiment. This analysis was used as a loading control to ensure equivalent amounts of plasmids were loaded on gels for Western blot. (G) SJG-136 treated plasmids were incubated in HSS, and soluble extracts were analyzed by Western blot.
	Figure S2. MutSa and PCNA mutants cloned and purified, Related to Figure 2 (A) Schematic of X.l. MSH6. The domain structure of this protein and the locations of mutations used in the study are illustrated. (B) The sequence of the conserved FXE domain in MSH6 is illustrated for several species. The amino acid substitutions used in this study are indicated in red. (C) The sequence of the conserved PIP domain in MSH6 is illustrated for several species. The triple amino acid substitution used in this study is indicated in red. (D) Purified MutSaWT, MutSaFXE, or MutSaPIP complex stained with Coomassie blue. (E) The sequence of the conserved interdomain connector loop of PCNA is illustrated for several species. The double amino acid substitution used in this study is indicated in red. (F) Purified PCNAWT and PCNAL126A,I128A stained with Coomassie blue. (G) Co-immunoprecipitation of purified recombinant WT and mutant MSH2-MSH6 and PCNA using preimmune and purified IgG- or MSH2-specific antibodies. Immunoprecipitates were analyzed by Western blot with the indicated antibodies (left panel). The combination of MSH2-MSH6 and PCNA mutants analyzed are indicated in the table (right panel). (H) PCNA immunodepletion. Mock- and PCNA-depleted HSS was analyzed by Western blot with the indicated antibodies to demonstrate depletion of PCNA. (I) Quantification of ICL repair in Mock-, PCNA-depleted, or PCNA-depleted HSS supplemented with recombinant PCNAWT, or PCNAL126A,I128A at 3 hrs. Results represent mean ± SEM of n=4 independent experiments.
	Figure S3. Nicking sites on the ICL plasmid and purification of MutLa, Related to Figure 3 (A) Sequence surrounding the trimethylene GpC-ICL lesion, and the specific locations of near (top) and far (bottom) nicking sites used in Figure 3A,B. (B) Schematic of X.l. PMS2. The domain structure of this protein and the locations of mutations used in the study are illustrated. (C) The sequence of the conserved catalytic nuclease domain in PMS2 is illustrated for several species. The amino acid substitution used in this study is indicated in red. (D) Purified MutLaWT and MutLan.d. complex stained with Coomassie blue. (E) Plasmid pull-down assay of SJG-136 treated plasmids in MLH1-depleted extract (left), or HSS supplemented with MutLaWT and MutLan.d. (right). Plasmid-bound proteins were analyzed by Western blot.
	Figure S4. RPA is required for RIR, Related to Figure 4 (A) Schematic of X.l. EXO1. The domain structure of this protein and the locations of mutations used in the study are illustrated. (B) Control or SJG-136 ICL oligonucleotide duplexes run on a denaturing polyacrylamide gel and stained with SYBR Gold. (C) RPA immunodepletion. Mock- and RPA-depleted HSS was analyzed by Western blot to demonstrate depletion of RPA. (D) Quantification of ICL repair in Mock-, RPA-depleted, or RPA-depleted HSS supplemented with recombinant RPAWT at 3 hrs. Results represent mean ± SEM of n=4 independent experiments. (E) Purified RPAWT stained with Coomassie blue. (F) Plasmid pull-down assay of SJG-136 treated plasmids in Mock- or RPA-depleted HSS. Plasmid bound proteins were analyzed by Western blot using the indicated antibodies.
	Figure S5. Sequences of site specific trimethylene and NM-like ICL plasmids, Related to Figure 5 (A) Sequence of oligonucleotide duplexes with a single trimethylene 5’ GpC-, 5’ CpG-, or NM-like ICLs. (B) Schematic of plasmids containing single site-specific trimethylene 5’ GpC- or 5’ CpG-ICL. Plasmids were generated by ligating trimethylene oligonucleotides in (A) into pEGFP. ICL repair was measured using qPCR with X and C primers as indicated.

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