Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities

Abstract

Rrm3p is a 5-to-3 DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.

Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

“…Since deletion of ScPIF1 leads to an increase in mitochondrial DNA point mutations, particularly after oxidative damage (21,51,52,67), it is also possible mPif1 plays a nonessential role in mitochondrial genome stability. Although Scrrm3⌬ cells do not exhibit changes in GCR or mitochondrial genome stability, these cells do exhibit genetic instability, presumably due to replication fork pausing at specific chromosomal loci, such as the ribosomal DNA locus (31,32,45,59,68,69). ScRRM3 and ScPIF1 exhibit genetic interactions (including synthetic lethality) with many gene products required in DNA replication and S-phase checkpoint arrest (1,2,17,50,58,59,69,71).…”

“…Although Scrrm3⌬ cells do not exhibit changes in GCR or mitochondrial genome stability, these cells do exhibit genetic instability, presumably due to replication fork pausing at specific chromosomal loci, such as the ribosomal DNA locus (31,32,45,59,68,69). ScRRM3 and ScPIF1 exhibit genetic interactions (including synthetic lethality) with many gene products required in DNA replication and S-phase checkpoint arrest (1,2,17,50,58,59,69,71). Thus, additional inactivation of other DNA helicases involved in DNA replication or repair or proteins essential for the S-phase checkpoint may yet reveal an interesting phenotype in mPif Ϫ/Ϫ animals.…”

“…Unlike Rrm3 and Pif1, Pfh1 is essential (77). Rrm3 promotes the progression of replication forks at particular chromosomal loci prone to pausing during DNA replication (including the telomere), and rrm3⌬ cells exhibit a modest telomere lengthening (11,32,33,45,59,69). Mutation of ScPif1 leads to telomerase-dependent telomere lengthening (33,76), and this phenotype depends on the ATPunwinding activity of ScPif1; the enzyme has a preference for short (Ͻ30 nucleotides) RNA-DNA hybrids and can unwind telomerase RNA from a telomeric DNA substrate (15).…”

The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

“…In the absence of Rrm3, over 1,000 natural protein-DNA RF pauses throughout the genome become markedly enhanced, leading to increased RF breakage 10,13 . Consistent with RF pausing at protein-DNA complexes being a universal threat to genome stability, Escherichia coli also possesses accessory DNA helicases that act to clear protein-DNA barriers ahead of RFs 14 .…”

Pictures, progress, and perplexities: The immediate cell biological effects of ionizing radiation

“…The unique role of the Mre11 complex as a "molecular clothespin" therefore enables it to participate in diverse cellular functions, including the stabilization of replication forks during S phase, during which Mre11 foci are detectable even in the absence of DNA damage. 51,52 The tethering of DNA strands may not be an initial event in DNA damage recognition, which is likely why there is not an absolute colocalization between γ-H2AX and Mre11 foci immediately after IR.…”