Peterson SE , Li Y , Chait BT , Gottesman ME , Baer R , Gautier J .

R01CA092245 NCI NIH HHS , R01GM077495 NIGMS NIH HHS , RR00862 NCRR NIH HHS , RR022220 NCRR NIH HHS , P01 CA097403 NCI NIH HHS , R01 GM077495 NIGMS NIH HHS , R01 CA092245 NCI NIH HHS , P41 RR000862 NCRR NIH HHS , U54 RR022220 NCRR NIH HHS

	Figure 1. DSB resection in S-phase Xenopus cell-free extract proceeds by CtIP-dependent and -independent pathways. (A) Kinetics of recruitment of proteins to DSB-containing chromatin in S-phase extract. S-phase extract was preincubated with sperm chromatin (5,000 sperm/µl). Aliquots of the sample were taken before (0 min) and at the indicated time (minutes) after addition of 0.05 U/µl PflMI restriction endonuclease. At each time point, 0.5 µl of the sample was removed and processed for Western blotting (extract), and 15 µl of the sample was removed and processed for chromatin isolation followed by Western blotting with the indicated antibodies (chromatin). A sample with no chromatin added (no sperm) serves as a chromatin purification control in A–C. (B) Early resection is dependent on MRN and CtIP in S-phase extract. Short kinetics of protein recruitment to chromatin in response to DSBs in mock-depleted, Mre11-depleted, and CtIP-depleted S-phase extracts as in A. (C) An MRN-CtIP–independent resection pathway operates in S-phase extract. Mock-depleted or CtIP-depleted S-phase extract was incubated with sperm chromatin and either buffer or PflMI restriction endonuclease for extended time points as in A. (D) Addition of recombinant xCtIP protein to CtIP-depleted S-phase extract restores resection activity to control levels. Experiment performed as in A but with an S-phase extract that was mock depleted, CtIP depleted supplemented with 110 nM recombinant xCtIP protein, or CtIP depleted as indicated. (E) The CtIP–BRCA1 interaction is not required for resection of endonuclease-induced chromosomal DSBs. Sperm chromatin was incubated in S-phase extract that was mock depleted, CtIP depleted, or CtIP depleted supplemented with 75 nM wt or S328A-xCtIP and was treated with buffer (−) or PflMI (+) for 30 min. Samples were processed for chromatin isolation as in A. The black line indicates that intervening lanes have been spliced out.
	Figure 2. Characteristics of CtIP-independent resection. (A) Recruitment of late resection pathway components in the absence of CtIP. The mock- and CtIP-depleted S-phase extract was treated with PflMI restriction endonuclease, and chromatin was isolated at the indicated time points (minutes) followed by Western blotting with the indicated antibodies as in Fig. 1 A. (B) CtIP-independent resection does not occur in membrane-free HSS extract. Mock-depleted or CtIP-depleted membrane-free S-phase HSS extract was preincubated with sperm chromatin (5,000 sperm/µl). Aliquots were taken before (0 min) and at the indicated time after addition of PflMI restriction endonuclease (0.05 U/µl) and processed as in Fig. 1 A. (C) CtIP-independent resection does not require DNA replication. CtIP-depleted S-phase extract was supplemented with recombinant geminin protein, roscovitine (roscov.), both, or buffer and was preincubated with sperm chromatin. (top) 15-µl aliquots of the sample were taken before (0 min) and at the indicated times after addition of 0.05 U/µl PflMI restriction enzyme and processed for chromatin isolation followed by Western blotting. (bottom) In parallel, a 10-µl aliquot of each sample was taken before addition of the restriction enzyme supplemented with 0.1 µl [32P]deoxy-CTP (dCTP) and incubated for 30 min to monitor DNA replication.
	Figure 3. DSBs generated in M phase undergo only CtIP-dependent resection. (A) Kinetics of recruitment of proteins to DSB-containing chromatin in M-phase extract. M-phase extract (meiotic and CSF arrested) was preincubated with sperm chromatin (5,000 sperm/µl). 15-µl aliquots of the sample were taken before (0 min) and at the indicated times (minutes) after addition of PflMI restriction endonuclease (0.05 U/µl) and processed for chromatin isolation followed by Western blotting with the indicated antibodies as in Fig. 1 A. (B) All resection is dependent on MRN and CtIP in M-phase extract. Short kinetics of protein recruitment to chromatin in response to DSBs in mock-depleted, Mre11-depleted, and CtIP-depleted M-phase extracts as in Fig. 1 B. (C) No resection occurs in the absence of CtIP in the M-phase extract. Long kinetics of protein recruitment to chromatin in response to DSBs. The mock-depleted or CtIP-depleted M-phase extract was incubated with sperm chromatin and either buffer or PflMI restriction endonuclease for extended time points as in Fig. 1 C. (D) Addition of recombinant xCtIP protein to CtIP-depleted M-phase extract restores resection activity to control levels. Experiment performed as in Fig. 1 D but with the M-phase extract that was mock depleted (+), CtIP depleted (Δ), or CtIP depleted supplemented with 50 nM recombinant xCtIP protein (Δ with xCtIP protein above) as indicated for a 30-min time point. The black line indicates that intervening lanes have been spliced out.
	Figure 4. DSB resection occurs in cycling mitotic Xenopus extract and mitosis of human cells. (A) Schematic timeline of the cycling mitotic extract experiment. Cycling extract was incubated with sperm chromatin (5,000 sperm/µl). Nocodazole was added at 96 min to trap the nuclei in the subsequent mitosis. Microscopy analysis 34 min later confirmed that the chromatin was in a highly condensed state indicative of mitosis. An aliquot was taken before addition of 0.05 U/µl PflMI restriction endonuclease at 163 min (time 0). Aliquots were also taken at 10, 30, and 60 min after addition of PflMI and processed for chromatin isolation and Western blotting. (B) Resection of DSBs occurs in nocodazole-arrested mitotic extract. After arrest with nocodazole, DSBs were induced in the mitotic chromatin, and aliquots were taken before and at the indicated time points (minutes) after addition of PflMI and processed for chromatin isolation and Western blotting with the indicated antibodies. The mitotic status of the extract was also confirmed by the presence of pSer10–histone H3. (C) Resection of DSBs occurs in mitosis of human cells. Asynchronous HeLa cells were grown on 8-well chamber slides and mock irradiated (no damage) or microirradiated using a high-energy UV laser microscope (PALM MicroBeam IV). After the indicated time (±4 min), slides were processed and stained with antibodies against human RPA34 and phospho–Ser10-histone H3. Bar, 10 µm.
	Figure 5. Phosphorylation of CtIP by Cdk1 is required for M-phase resection. (A) Cdk activity is required for CtIP function and M-phase resection. M-phase extract was treated with DMSO, 200 µM roscovitine (Rosc.), 200 µM RO-3306, or both, and chromatin was isolated at the indicated time points (minutes) after addition of PflMI restriction endonuclease. Quantification of relative CtIP chromatin binding from three independent experiments is shown on the bottom, with error bars representing one standard deviation. (B) Cdk1–cyclin B kinase phosphorylates recombinant xCtIP in vitro. Recombinant Cdk1–cyclin B was incubated with recombinant xCtIP (wt or T806A) as indicated in the presence of γ-[32P]ATP. Samples were analyzed by SDS-PAGE and autoradiography. Quantification of the autoradiograph signal for xCtIP in this experiment is graphed on the bottom. Note that xCtIP-T806A incorporation is reduced by ∼25% because of the presence of multiple Cdk phosphorylation sites. (C) Tandem mass spectrometry of endogenous M-phase CtIP reveals phosphorylation at S805/T806. Tandem mass spectrum of phosphopeptide spanning residues 801–822. The experimental molecular mass of the intact precursor ion (2664.1613 D) closely matches the theoretical mass of the tryptic peptide 801–822 plus a phosphate group (2664.1607 D; mass difference = 0.0006 D). The fragmentation pattern confirms the identity of this phosphopeptide and narrows the localization of the phosphate group to either S805 or T806. The asterisk indicates that the cysteine residue was alkylated using iodoacetamide. (D) Conserved residue T806 is required for CtIP activity and resection in M phase. M-phase extract was mock depleted or CtIP depleted. CtIP-depleted extract was supplemented with buffer (−), wt xCtIP (wt), xCtIP-T806A (T806A), or xCtIP-T806E (T806E). PflMI restriction endonuclease (+) or buffer (−) was added, and chromatin was isolated after 15 min. Time points above are in minutes. m/z, mass to charge ratio.
	Figure 6. M-phase Cdk1 activity inhibits Rad51 loading on ssDNA-RPA. (A) Rad51 accumulates on resected chromatin, and Chk1 becomes activated in S- but not in M-phase extract. S- and M-phase extracts prepared from the same batch of eggs were either mock or CtIP depleted. Chromatin-binding time course in response to DSBs was performed as in Fig. 1 A. (B) Treatment of S-phase extract with Cdk1 inhibits Rad51 chromatin binding but not Chk1 activation. S-phase extract was supplemented with 100 nM recombinant Cdk1–cyclin B protein complex or buffer, and a chromatin-binding time course in response to DSBs was performed as in Fig. 1 A. (C) Inhibition of Cdk1 activity in M phase restores Rad51 accumulation in response to chromosomal DSBs. M-phase extract was supplemented with the specific Cdk1 inhibitor RO-3306 (200 µM) or DMSO, and a chromatin-binding time course in response to DSBs was performed as in Fig. 1 A. Time points above the blots are in minutes.
	Figure 7. Chromosomal DNA DSB resection in M phase. DNA DSBs generated in S phase are resected via the concerted action of MRN-CtIP and two additional pathways represented here as a single Exo1-DNA2 entity. S-phase resection generates ssDNA-RPA, which is competent for Rad51 filament assembly, a necessary step for HR. In contrast, DSBs generated in M phase are processed into ssDNA-RPA intermediates that do not support Rad51 chromatin assembly. Cdk1 promotes resection by phosphorylating CtIP while at the same time inhibiting Rad51 chromatin assembly. Resection initiation is dependent on MRN-CtIP in M phase as reflected by the relative size of the resection machinery components. M-phase resection generates ends that are not compatible for repair by NHEJ or by HR. These ends could be substrates for microhomology-mediated end joining in M or G1 phase. Alternatively, they could be transmitted to the next S phase and repaired by HR. P, phosphorylation.

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