Role of 53BP1 in end protection and DNA synthesis at DNA breaks

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.

Marker-free coselection for successive rounds of prime editing in human cells

“…17 ). This spectrum of byproducts is dissimilar to the ∼1-5bp indels typically observed with Cas9 nucleases and has been previously reported 59 . In fact, most clones (55/58) that were not homozygous for the PE3-specified MTOR mutations contained undesired insertions, deletions or pegRNA scaffold incorporations ( Table 1 and Supplementary Figs.…”

“…These editing byproducts are reminiscent to the DSB repair response resulting from presence of concomitant nicks 56 . Mechanistically, fill-in synthesis of the 3' protruding ends generated by concomitant nicks via Polα-primase could account for these tandem duplications in a pathway that requires 53BP1 [57][58][59] . We thus tested whether the inhibition of 53BP1 with i53 60 could prevent the formation of large PE3-mediated insertions by delaying rapid and predominant NHEJ-mediated repair.…”

Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks

“…In mammalian cells, it has been suggested that unrestricted resection may not be the only model for the increased ssDNA in the absence of 53BP1, rather it could also be due to impaired gap filling 9, 37 . Recent work has shown that 53BP1 facilitates gap filling by recruitment of Shieldin and subsequently the CST complex and Pol- alpha primase 38, 39 . However, there may be differences between the functions of Rad9 and 53BP1 as our data shows that gap filling occurs without delay in the absence of Rad9 and any role Rad9 may have in recruitment of gap filling factors is not essential for completion of fill-in synthesis.…”

The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability

“…CST facilitates RAD51 recruitment to forks, whereas RPA-coated ssDNA inhibits RAD51 nuclear filament formation [18]. During DSB repair, CST appears to promote c-NHEJ via inhibiting excessive end resection through a POLα-dependent fill-in mechanism [26,27,47], whereas RPA stimulates end resection and promotes homologous recombination [28][29][30]. The structural difference between CST and RPA may help in explaining distinct functionalities of these two ssDNA-binding complexes in various genome maintenance pathways.…”