Role of 53BP1 in end protection and DNA synthesis at DNA breaks
Open Access
- 9 September 2021
- journal article
- research article
- Published by Cold Spring Harbor Laboratory in Genes & Development
- Vol. 35 (19-20), 1356-1367
- https://doi.org/10.1101/gad.348667.121
Abstract
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 α (Polα) 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, Polα-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, Polα 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, Polα-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.Keywords
Funding Information
- National Institutes of Health (AI047829, AI074953)
- National Cancer Institute
- NIH (R01CA085344, R50CA211397)
- Intramural Research Program of the NIH
- Ellison Medical Foundation (Senior Scholar in Aging award, AG-SS-2633-11)
- Department of Defense (W81XWH-16-1-599, W81XWH-19-1-0652)
- Alex's Lemonade Stand Foundation
- NIH (Intramural FLEX Award)
This publication has 50 references indexed in Scilit:
- RIF1 Is Essential for 53BP1-Dependent Nonhomologous End Joining and Suppression of DNA Double-Strand Break ResectionMolecular Cell, 2013
- Controlling DNA-end resection: a new task for CDKsFrontiers in Genetics, 2013
- Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNAGenome Research, 2012
- DNA end resection by Dna2–Sgs1–RPA and its stimulation by Top3–Rmi1 and Mre11–Rad50–Xrs2Nature, 2010
- BigWig and BigBed: enabling browsing of large distributed datasetsBioinformatics, 2010
- 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancersNature Structural & Molecular Biology, 2010
- 53BP1 Inhibits Homologous Recombination in Brca1-Deficient Cells by Blocking Resection of DNA BreaksCell, 2010
- High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genomeThe EMBO Journal, 2010
- BEDTools: a flexible suite of utilities for comparing genomic featuresBioinformatics, 2010
- The Sequence Alignment/Map format and SAMtoolsBioinformatics, 2009