Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue

Abstract

The disease-associated nuclease–helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery.

DNA2 in Chromosome Stability and Cell Survival—Is It All about Replication Forks?

“…According to model 1, Dna2 is indispensable for lagging strand replication, specifically for the removal of DNA flaps at maturing Okazaki fragments [ 29 ]. Model 2 suggests that Dna2 is essential for proper processing and recovery of stalled RFs [ 30 ]. These models, which are largely based on work in yeast, provide a framework to rationalize the most severe cellular and organismal phenotypes associated with DNA2 dysfunction.…”

“…However, recent findings indicating that, in dna2 Δ cells, Pif1-dependent toxicity arises at unresolved RFs as a result of excessive recombination-dependent replication (RDR) restart, suggest otherwise [ 84 ]. This link between failed RF recovery in dna2 Δ cells and Pif1-driven cell death, as explained in more detail below, indicates that Dna2-dependent RF recovery represents not only an important function in promoting replication completion, but also the sought-after essential function provided by Dna2 to ensure cell survival [ 30 , 84 ].…”

“…DNA synthesis in the context of a D-loop can proceed over long distances, but D-loop collapse is frequent, resulting in the exposure of long tracts of newly synthesized ssDNA [ 95 , 96 , 97 , 98 , 99 ]. Accordingly, excessive use of RDR in lieu of RF recovery by Dna2 could create a situation where toxic levels of ssDNA are generated through D-loop collapse, providing a plausible explanation for Rad9-dependent checkpoint activation and terminal cell-cycle arrest in Dna2-mutant cells [ 30 , 84 ] ( Figure 4 ). In contrast to OFP, where nascent nucleosomes and flap formation restrict any extended strand-displacement DNA synthesis by Pol δ [ 32 , 40 , 60 , 62 ], RDR DNA-synthesis tracts can be thousands of nucleotides in length.…”

“…As described in Section 2 , yeast Dna2 has been implicated in DSB end-resection, OFP, and RF recovery. The latter two pathways have each been proposed to account for the essential requirement for DNA2 [ 29 , 30 ]. Precise measurements of Okazaki fragment maturation in vitro, and recent Okazaki fragment sequencing results in particular, have indicated that the contribution of Dna2 to OFP could be much less significant than previously assumed [ 40 , 61 ].…”

“…The literature to date is consistent with the conjecture that the essential role of human DNA2 is intimately linked to the recovery of stalled RFs, reflecting what has been suggested in yeast [ 84 ]. Whether, as proposed in yeast, there is an additional risk of alternative RF restart by unscheduled RDR or other pathways associated with checkpoint activation and cell-cycle arrest remains to be addressed in human cells [ 30 , 84 ].…”