Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions

Abstract

The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11/CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN/Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11/CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11/CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

“…Moreover, it has been demonstrated that WRN depletion leads to a marked reduction in the frequency of RPA and BrdU/ssDNA foci formed in response to ionizing radiation, indicative of a resection defect (52). A similar phenotype has been observed in DNA2-depleted cells (18).…”

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

“…In line with this hypothesis, HP1α knockdown impairs both 53BP1 and BRCA1 recruitment to sites of damage. 17 Given that 53BP1 and BRCA1 have opposite effects on DNA-end resection, [30][31][32][33] further research on this particular issue would certainly be critical to shed light on the molecular details of HP1 role during this process.…”

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

“…To specifically monitor hExo1-dependent resection, we first measured resection in DNA2-depleted cells. DNA2-depleted cells displayed only a minor DNA resection defect (11,12). To define the role of hRPA on hExo1 resection, we next quantified DNA resection intermediates in cells that were depleted for both RPA2 and DNA2.…”

“…In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8,9). Exo1 also promotes DSB repair via homologous recombination (HR) by processing the free DNA ends to generate kilobase-length ssDNA resection products (1,(10)(11)(12). The resulting ssDNA is paired with a homologous DNA sequence located on a sister chromatid, and the missing genetic information is then restored via DNA synthesis.…”