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.

Keywords: Chromosome stability; DNA replication fork; DNA replication stress; DNA2 nuclease–helicase; Homologous recombination; Seckel syndrome.

Fig. 1

Processing and restart of stalled RFs. a When a stalled RF cannot easily resume DNA synthesis, it may backtrack into a reversed position, forming a four-way DNA junction. Reversed RFs have emerged as key intermediates of RF recovery, facilitating passive rescue by fork convergence or further processing to restore RF activity. b Breakage of stalled and reversed RFs can occur through attack by structure-specific nucleases (black arrowheads). The resulting single-ended DNA double-strand break undergoes DNA end-resection and Rad51-mediated strand invasion to initiate HR-dependent replication restart by BIR. c HR-dependent replication restart can also occur at unbroken reversed RFs. End-resection at the regressed branch produces the 3′-DNA overhang for Rad51-mediated strand-invasion, restarting replication along the RDR pathway

Fig. 2

Dna2 is an essential gatekeeper to HR-dependent restart of stalled replication forks. By degrading dissociated nascent DNA at stalled RFs, Dna2 counteracts RF reversal, promoting the resumption of DNA synthesis and/or RF convergence to mediate complete genome replication. By processing stalled RFs, Dna2 also limits opportunities for RF restart by RDR, and this makes Dna2 indispensable for cell survival. While RDR provides an important salvage pathway for stalled RFs, DNA synthesis in the context of a displacement loop (D-loop) is unstable. In the absence of Dna2, excessive RDR results in an accumulation of unsustainably high levels of recombination by-products by way of D-loop collapse. Exposure of long ssDNA tracts then causes Rad9-dependent checkpoint activation and terminal cell-cycle arrest. For further details, see main text