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, ATPdependent, 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.

During each yeast cell cycle, ∼100,000 nicks are generated during lagging-strand DNA replication. Efficient nick processing during Okazaki fragment maturation requires the coordinated action of DNA polymerase ␦ (Pol ␦) and the FLAP endonuclease FEN1. Misregulation of this process leads to the accumulation of double-stranded breaks and cell lethality. Our studies highlight a remarkably efficient mechanism for Okazaki fragment maturation in which Pol ␦ by default displaces 2-3 nt of any downstream RNA or DNA it encounters. In the presence of FEN1, efficient nick translation ensues, whereby a mixture of mono-and small oligonucleotides are released. If FEN1 is absent or not optimally functional, the ability of Pol ␦ to back up via its 3-5-exonuclease activity, a process called idling, maintains the polymerase at a position that is ideal either for ligation (in case of a DNA-DNA nick) or for subsequent engagement by FEN1 (in case of a DNA-RNA nick). Consistent with the hypothesis that DNA polymerase is the leading-strand enzyme, we observed no idling by this enzyme and no cooperation with FEN1 for creating a ligatable nick. The nature of semidiscontinuous DNA replication in a eukaryotic cell mandates the synthesis of short RNAprimed Okazaki fragments on the lagging strand. During each cell cycle, tens of millions of these small Okazaki fragments are synthesized in a mammalian cell. Even during replication of the much more compact yeast genome, an estimated ∼100,000 Okazaki fragments are synthesized. Each of these fragments needs to be matured into continuous double-stranded DNA with high fidelity. Unrepaired nicks lead to double-strand breaks during the next cell cycle. These breaks can be repaired by the double-strand break repair system; however, the accumulation of as few as 30 unsealed nicks can overwhelm this system in yeast and cause cell lethality (Resnick 1978). Therefore, it is essential not only that the maturation of Okazaki fragments occurs with high speed and high fidelity, but also that backup mechanisms are available for the infrequent occasions that the prevailing maturation machinery fails.Several different models have been proposed to account for initiator RNA degradation and gap filling during the maturation of Okazaki fragments (for review, see Kao and Bambara 2003). In one proposed mechanism, the initiator RNA is degraded by the sequential action of RNase H2 and the FLAP endonuclease FEN1. A second mechanism has been proposed in which the initiator RNA is degraded by the sequential action of the nuclease/helicase Dna2 and FEN1. Based on biochemical studies, we, and others, have recently proposed a third model for Okazaki fragment maturation, in which a simple machinery of DNA polymerase ␦ (Pol ␦) and FEN1 carries out nick translation in a processive complex with the replication clamp PCNA (proliferating cell nuclear antigen) in order to degrade the RNA primer (Maga et al.

Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.

BackgroundG-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo.ResultsWe determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites.ConclusionsIn the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0101-5) contains supplementary material, which is available to authorized users.