DNA2 and EXO1 in replication-coupled, homology-directed repair and in the interplay between HDR and the FA/BRCA network

Abstract

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.

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

“…A similar phenotype has been observed in DNA2-depleted cells (18). Although the previous studies did not address the relationship between WRN and DNA2, they demonstrated that these enzymes act synergistically with EXO1 to promote DNA end resection in human cells (18,52). A role for WRN as a critical DNA end resection factor is also consistent with the cellular phenotype of Werner syndrome, a severe premature aging disorder caused by inherited mutations in the WRN gene (53).…”

“…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). Although the previous studies did not address the relationship between WRN and DNA2, they demonstrated that these enzymes act synergistically with EXO1 to promote DNA end resection in human cells (18,52).…”

“…The molecular machinery of DNA end resection appears to be largely conserved between yeast and man (15)(16)(17)(18)(19). However, it remains a matter of debate which DNA helicase mediates DNA2-catalyzed resection in mammalian cells.…”

To trim or not to trim: Progression and control of DSB end resection

“…50,55 Simultaneous depletion of BLM and EXO1 in U2OS cells treated with CPT significantly impairs RPA phosphorylation and focal accumulation, as well as the consequent ATR-mediated signaling. 50 Human DNA2 facilitates repair of replication-associated DSBs by promoting ssDNA formation at stalled replication forks, 56,57 acting redundantly with hEXO1. 48,57 In vitro data confirm that end resection occurs through two pathways, suggesting that the relationship between BLM-DNA2 and hEXO1 is not simply complementary.…”

Novel Pathways Linking Mammalian Septins and the DNA Damage Response

“…DNA2 and EXO1 have been shown to be important in end resection during DSB repair [195,197]. Interestingly, although DNA2 and EXO1, working in concert, are necessary for DNA end resection, their depletion results in a defect in SSA and an increase in HR [345]. Thus, it appears the role of these nucleases during end resection alters the balance between which repair pathway is utilized.…”