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.