PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes.
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ABSTRACT:
SUBMITTER: Tran PLT
PROVIDER: S-EPMC5413955 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Saccharomyces cerevisiae expresses two Pif1-family helicases-Pif1 and Rrm3-which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterized the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulates strand displacement by DNA polymerase ? during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication-fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes. In agreement with this observation, we found that head-on collisions between tRNA transcription and replication are under-represented in the S. cerevisiae genome. We demonstrate that tRNA-mediated arrest is R-loop independent and propose that replisome arrest and DNA damage are mechanistically separable.
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Project description:The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans. Here we show that exceptionally potent G-quadruplex unwinding is conserved among Pif1 helicases. Moreover, Pif1 helicases from organisms separated by more than 3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast. The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to new genetic and epigenetic changes. Furthermore, when expressed in yeast, human PIF1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.
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Project description:Saccharomyces cerevisiae encodes two distinct Pif1-family helicases – Pif1 and Rrm3 – which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging- strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes; consistent with this observation, we find that head-on collisions between tRNA transcription and replisome progression are under-represented in the S. cerevisiae genome. Further, we demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable.
2016-10-17 | GSE71973 | GEO
Project description:Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication. We show that loss of RRM3 results in elevated spontaneous mutations made by DNA polymerases Pols ϵ and δ, which are subject to DNA mismatch repair. The absence of RRM3 also causes higher mutagenesis by the fourth B-family DNA polymerase Pol ζ. By genome-wide analysis, we show that the mutational consequences due to loss of RRM3 vary depending on the genomic locus. Rrm3 promotes the accuracy of DNA replication by Pols ϵ and δ across the genome, and it is particularly important for preventing Pol ζ-dependent mutagenesis at tRNA genes. In addition, mutation avoidance by Rrm3 depends on its helicase activity, and Pif1 serves as a backup for Rrm3 in suppressing mutagenesis. We present evidence that the sole human Pif1 family helicase in human cells likely also promotes replication fidelity, suggesting that a role for Pif1 family helicases in mutation avoidance may be evolutionarily conserved, a possible underlying mechanism for its potential tumor-suppressor function.
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Project description:Members of the Pif1 family of helicases function in multiple pathways that involve DNA synthesis: DNA replication across G-quadruplexes; break-induced replication; and processing of long flaps during Okazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNA replication across arrays of proteins tightly bound to DNA. This is a surprising feat since DNA rewinding or annealing activities limit the amount of single-stranded DNA product that Pif1 can generate, leading to an apparently poorly processive helicase. In this work, using single-molecule Förster resonance energy transfer approaches, we show that 2 members of the Pif1 family of helicases, Pif1 from Saccharomyces cerevisiae and Pfh1 from Schizosaccharomyces pombe, unwind double-stranded DNA by a branched mechanism with 2 modes of activity. In the dominant mode, only short stretches of DNA can be processively and repetitively opened, with reclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches of DNA are unwound via a path that is separate from the one leading to repetitive unwinding. Analysis of the kinetic partitioning between the 2 different modes suggests that the branching point in the mechanism is established by conformational selection, controlled by the interaction of the helicase with the 3' nontranslocating strand. The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.
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Project description:The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms.
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Project description:Helicases are ubiquitous enzymes found in all organisms that are necessary for all (or virtually all) aspects of nucleic acid metabolism. The Pif1 helicase family is a group of 5'-->3' directed, ATP-dependent, super family IB helicases found in nearly all eukaryotes. Here, we review the discovery, evolution, and what is currently known about these enzymes in Saccharomyces cerevisiae (ScPif1 and ScRrm3), Schizosaccharomyces pombe (SpPfh1), Trypanosoma brucei (TbPIF1, 2, 5, and 8), mice (mPif1), and humans (hPif1). Pif1 helicases variously affect telomeric, ribosomal, and mitochondrial DNA replication, as well as Okazaki fragment maturation, and in at least some cases affect these processes by using their helicase activity to disrupt stable nucleoprotein complexes. While the functions of these enzymes vary within and between organisms, it is evident that Pif1 family helicases are crucial for both nuclear and mitochondrial genome maintenance.
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Project description:Pif1 family helicases are found in virtually all eukaryotes. Saccharomyces cerevisiae (Sc) encodes two Pif1 family helicases, ScPif1 and Rrm3 ScPif1 is multifunctional, required not only for maintenance of mitochondrial DNA but also for multiple distinct nuclear functions. Rrm3 moves with the replication fork and promotes movement of the fork through ∼1400 hard-to-replicate sites, including centromeres. Here we show that ScPif1, like Rrm3, bound robustly to yeast centromeres but only if the centromere was active. While Rrm3 binding to centromeres occurred in early to mid S phase, about the same time as centromere replication, ScPif1 binding occurred later in the cell cycle when replication of most centromeres is complete. However, the timing of Rrm3 and ScPif1 centromere binding was altered by the absence of the other helicase, such that Rrm3 centromere binding occurred later in pif1-m2 cells and ScPif1 centromere binding occurred earlier in rrm3Δ cells. As shown previously, the modest pausing of replication forks at centromeres seen in wild-type cells was increased in the absence of Rrm3 While a lack of ScPif1 did not result in increased fork pausing at centromeres, pausing was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Likewise, centromere function as monitored by the loss rate of a centromere plasmid was increased in rrm3Δ but not pif1Δ cells, and was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Thus, ScPif1 promotes centromere replication and segregation, but only in the absence of Rrm3 These data also hint at a potential post-S phase function for ScPif1 at centromeres. These studies add to the growing list of ScPif1 functions that promote chromosome stability.
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Project description:The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and mutations in three of these, BLM, WRN, and RECQL4, are associated with human diseases and a cellular phenotype that includes genomic instability. To date, no human disease has been associated with mutations in RECQL and no cellular phenotype has been associated with its deficiency. To gain insight into the physiological function of RECQL, we disrupted Recql in mice. RECQL-deficient mice did not exhibit any apparent phenotypic differences compared to wild-type mice. Cytogenetic analyses of embryonic fibroblasts from the RECQL-deficient mice revealed aneuploidy, spontaneous chromosomal breakage, and frequent translocation events. In addition, the RECQL-deficient cells were hypersensitive to ionizing radiation, exhibited an increased load of DNA damage, and displayed elevated spontaneous sister chromatid exchanges. These results provide evidence that RECQL has a unique cellular role in the DNA repair processes required for genomic integrity. Genetic background, functional redundancy, and perhaps other factors may protect the unstressed mouse from the types of abnormalities that might be expected from the severe chromosomal aberrations detected at the cellular level.
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Project description:Replication stress activates the Mec1(ATR) and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1.
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