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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. 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

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