Role Of 53bp1 In End Protection And Dna Synthesis At Dna Breaks

In this study, Paiano et al. examine the mechanism by which 53BP1 counteracts end resection at DNA double-strand breaks (DSBs). Using genetic and genomics approaches, the authors show that, in the presence of 53BP1, Pol alpha-dependent synthesis counteracts resection at DSBs. However, in the absence of 53BP1, Pol alpha is surprisingly still active at DSBs but is unable to counteract hyperresection of DNA ends. The authors conclude that the end protection function of 53BP1 is likely to be the predominant mechanism by which it blocks end resection.Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase alpha (Pol alpha) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Pol alpha-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Pol alpha activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Pol alpha-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.