Abstract
DNA2 is an essential nuclease–helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5′-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5′-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2’s role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1’s ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2’s role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.
Highlights
Dna2 is a conserved enzyme of DNA metabolism that comprises a nuclease domain with single-stranded DNA-specific endonuclease activity fused to a superfamily 1 helicase domain with 5 -to-3 translocation polarity [1,2,3]
To address the impact of absent Dna2 on DNA replication, we characterized dna2 cells whose viability was restored by replacing PIF1 with the pif1-m2 allele [14]
Ethidium bromide staining after Pulsed field gel electrophoresis (PFGE) conspicuously showed a weaker increase of gelresolved chromosomal DNA for dna2Δ pif1-m2 cells passing through S phase compared to pif1-m2 control cells in the region of chromosome XII (Figure 1A)
Summary
Dna is a conserved enzyme of DNA metabolism that comprises a nuclease domain with single-stranded DNA (ssDNA)-specific endonuclease activity fused to a superfamily 1 helicase domain with 5 -to-3 translocation polarity [1,2,3]. DNA2 is essential for cell proliferation and embryonic development across species [2,4,5,6]. DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing, telomere homeostasis, centromeric DNA replication, and the recovery of stalled replication forks (RFs) [13]. Pinpointing which of its functions are critical for cell proliferation is an important step in rationalizing the molecular pathologies associated with DNA2
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