A mechanism for the suppression of homologous recombination in G1 cells

Abstract

DNA repair by homologous recombination (HR)1 is highly suppressed in G1 cells2,3 to ensure that mitotic recombination occurs solely between sister chromatids4. Although many HR factors are cell cycle-regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 in order to constrain BRCA2 function to the S/G2 phases. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein5, in complex with CUL3-RBX16. PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA end resection is sufficient to induce HR in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR/Cas9-based gene targeting assay. We conclude that the mechanism prohibiting HR in G1 minimally consists of the suppression of DNA end resection coupled to a multi-step block to BRCA2 recruitment to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce HR in G1 cells with defined factors could spur the development of gene targeting applications in non-dividing cells.

Keeping homologous recombination in check

“…Consequently, disruption of 53BP1 leads to the recruitment of BRCA1 to DSBs in G1 phase. In the recent Nature paper from Durocher's laboratory, Orthwein et al [2] discovered that although BRCA1 is localized to DSBs during G1 phase in 53BP1-deficient cells, it fails to recruit the BRCA2-PALB2 complex, which is consistent with the lack of HR activity in these cells.…”

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

“…36) However, the efficiency was not high possibly because of the nature of Cas9 that it generates DSB with blunt ends. DSB with blunt ends mainly allows homology-directed repairs (HDRs) which barely occur in non-dividing cells, 37) leading to the observed low efficiency. Conversely, in those cells, non-homologous end joining (NHEJ) is the main homologyindependent repair pathway where the alignment of only one to a few complementary bases at most are required for the religation of two ends.…”

“…Conversely, in those cells, non-homologous end joining (NHEJ) is the main homologyindependent repair pathway where the alignment of only one to a few complementary bases at most are required for the religation of two ends. 37) Therefore, the characteristics of Cpf1, that it cleaves genome with 5′-staggered ends, might facilitate HITI of donor fragments through sticky-ends. In fact, AsCpf1 and LbCpf1 have been shown recently to successfully perform correction of disease-related mutations in patient-derived induced pluripotent stem (iPS) cells via plasmid-electroporation as well as germline correction in the model mouse via mRNA-and gRNA-injection.…”

Genome engineering - Matching supply with demand

“…In contrast, HDR is only active in S and G2, and is suppressed in G1 due to the suppression of DNA-end resection coupled with a multistep block of BRCA2 to DNA damage sites. 1 A recent study refined this activity model further: cells in G1 repair DSBs exclusively by NHEJ. Cells then gradually increase their use of HDR as they progress from G1 to early S. The HDR activity peaks in mid-S and gradually declines as cells move toward late S and G2.…”