EXO1 suppresses double-strand break induced homologous recombination between diverged sequences in mammalian cells

Abstract

DNA double-strand breaks (DSBs) can be repaired through several mechanisms, including homologous recombination (HR). While HR between identical sequences is robust in mammalian cells, HR between diverged sequences is suppressed by DNA mismatch-repair (MMR) components such as MSH2. Exonuclease I (EXO1) interacts with the MMR machinery and has been proposed to act downstream of the mismatch recognition proteins in mismatch correction. EXO1 has also been shown to participate in extensive DSB end resection, an initial step in the HR pathway. To assess the contribution of EXO1 to HR in mammalian cells, DSB-inducible reporters were introduced into Exo1−/− mouse embryonic stem cells, including a novel GFP reporter containing several silent polymorphisms to monitor HR between diverged sequences. Compared to HR between identical sequences which was not clearly affected, HR between diverged sequences was substantially increased in Exo1−/− cells although to a lesser extent than seen in Msh2−/− cells. Thus, like canonical MMR proteins, EXO1 can restrain aberrant HR events between diverged sequence elements in the genome.

Functional deficiency of DNA repair gene EXO5 results in androgen-induced genomic instability and prostate tumorigenesis

“…We saw similar results in EXO1 knockout cells (Supplementary Fig. 9A), which is consistent with previous results showing that EXO1 knockout reduces HDR efficiency (35,36). However, knockout of the EXO5 gene in the LNCaP-NHEJ cell line, similar to knockout of EXO1 (37), did not significantly change NHEJ repair efficiency compared to that of the parental LNCaP-NHEJ cell line (Fig.…”

Exonuclease 1 (Exo1) Participates in Mammalian Non-Homologous End Joining and Contributes to Drug Resistance in Ovarian Cancer

“…Exo1 has been well characterized in end processing in MMR, HR, as well as nucleotide excision repair (NER) [39][40][41][42][43][44][45][46][47]. We identified Exo1 is necessary for efficient NHEJ, possibly via interaction with NHEJ required factor Ku70.…”

BLM has Contrary Effects on Repeat-Mediated Deletions, based on the Distance of DNA DSBs to a Repeat and Repeat Divergence

“…In contrast, EXO1 is important 0.3-3.5 kbp of end resection, based on this assay (Zhou et al, 2014). Nonetheless, EXO1 has been shown to be dispensable for homologous recombination events requiring this amount of end resection (i.e., <3 kbp; Chen et al, 2017;Rein et al, 2015). (A) Immunoblotting analysis for several mESC lines, and transfections of siRNA or complementation vectors, for RAD52, MSH2, BLM, and ACTIN.…”

“…Several mESC lines were previously described: WT J1 (Mendez-Dorantes et al, 2018), Blm tet/tet (Yusa et al, 2004), Msh2À/À (Claij and te Riele, 2004;Mendez-Dorantes et al, 2018), Exo1À/À (Chen et al, 2017), and Rad52À/À (Mendez-Dorantes et al, 2018). The Rad52À/ÀMsh2À/À mESC line was derived from the Rad52À/À cell line, using two Cas9-mediated DSBs to induce a deletion in Msh2 using two sgRNAs (Msh2 sgRNA 1 and 2, Table S1).…”

“…To examine EXO1, we integrated the RMD-GFP reporters in Exo1 À/À mESCs (Chen et al, 2017) and conducted the RMD assays along with an EXO1 complementation vector or control EV. With RMDs between identical repeats, Exo1 À/À versus WT mESCs showed a marked reduction in these events for long 3 0 -DSB/repeat distances (2.8-fold for 19 kbp and 3.8-fold for 28.4 kbp), which was reversed upon complementation with EXO1 (Figure 2D).…”