Quantitation of DNA double-strand break resection intermediates in human cells |
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| Authors: | Yi Zhou Pierre Caron Ga?lle Legube Tanya T Paull |
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| Institution: | 1.The Department of Molecular Biosciences, The Howard Hughes Medical Institute, The University of Texas at Austin, Austin, TX 78712, USA, 2.Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA, 3.Université de Toulouse, UPS, LBCMCP, 31062 Toulouse, France and 4.CNRS, LBCMCP, F-31062 Toulouse, France |
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| Abstract: | 5′ strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5′ strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle–dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection. |
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