XB-IMG-126408
Xenbase Image ID: 126408
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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. Image published in: Peterson SE et al. (2011) © 2011 Peterson et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |