Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA.

Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5'→3' direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA-binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA.

Role and Regulation of Pif1 Family Helicases at the Replication Fork.

Pif1 helicases are a multifunctional family of DNA helicases that are important for many aspects of genomic stability in the nucleus and mitochondria. Pif1 helicases are conserved from bacteria to humans. Pif1 helicases play multiple roles at the replication fork, including promoting replication through many barriers such as G-quadruplex DNA, the rDNA replication fork barrier, tRNA genes, and R-loops. Pif1 helicases also regulate telomerase and promote replication termination, Okazaki fragment maturation, and break-induced replication. This review highlights many of the roles and regulations of Pif1 at the replication fork that promote cellular health and viability.

Lysine acetylation regulates the activity of nuclear Pif1.

In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.

Structural study of the function of Candida Albicans Pif1.

Pif1 helicases, conserved in eukaryotes, are involved in maintaining genome stability in both the nucleus and mitochondria. Here, we report the crystal structure of a truncated Candida Albicans Pif1 (CaPif1368-883) in complex with ssDNA and an ATP analog. Our results show that the Q-motif is responsible for identifying adenine bases, and CaPif1 preferentially utilizes ATP/dATP during dsDNA unwinding. Although CaPif1 shares structural similarities with Saccharomyces cerevisiae Pif1, CaPif1 can contact the thymidine bases of DNA by hydrogen bonds, whereas ScPif1 cannot. More importantly, the crosslinking and mutant experiments have demonstrated that the conformational change of domain 2B is necessary for CaPif1 to unwind dsDNA. These findings contribute to further the understanding of the unwinding mechanism of Pif1.

Dynamic regulation of Pif1 acetylation is crucial to the maintenance of genome stability.

PIF1 family helicases are evolutionarily conserved among prokaryotes and eukaryotes. These enzymes function to support genome integrity by participating in multiple DNA transactions that can be broadly grouped into DNA replication, DNA repair, and telomere maintenance roles. However, the levels of PIF1 activity in cells must be carefully controlled, as Pif1 over-expression in Saccharomyces cerevisiae is toxic, and knockdown or over-expression of human PIF1 (hPIF1) supports cancer cell growth. This suggests that PIF1 family helicases must be subject to tight regulation in vivo to direct their activities to where and when they are needed, as well as to maintain those activities at proper homeostatic levels. Previous work shows that C-terminal phosphorylation of S. cerevisiae Pif1 regulates its telomere maintenance activity, and we recently identified that Pif1 is also regulated by lysine acetylation. The over-expression toxicity of Pif1 was exacerbated in cells lacking the Rpd3 lysine deacetylase, but mutation of the NuA4 lysine acetyltransferase subunit Esa1 ameliorated this toxicity. Using recombinant proteins, we found that acetylation stimulated the DNA binding affinity, ATPase activity, and DNA unwinding activities of Pif1. All three domains of the helicase were targets of acetylation in vitro, and multiple lines of evidence suggest that acetylation drives a conformational change in the N-terminal domain of Pif1 that impacts this stimulation. It is currently unclear what triggers lysine acetylation of Pif1 and how this modification impacts the many in vivo functions of the helicase, but future work promises to shed light on how this protein is tightly regulated within the cell.

Mms1 is an assistant for regulating G-quadruplex DNA structures.

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

Identification of Pif1 helicases with novel accessory domains in various amoebae.

Pif1 helicases are a conserved family of eukaryotic proteins involved in the maintenance of both nuclear and mitochondrial DNA. These enzymes possess a number of known and putative functions, which facilitate overall genome integrity. Here we have identified multiple subtypes of Pif1 proteins in various pathogenic and non-pathogenic amoeboid species which possess additional domains not present in other Pif1 helicases. These helicases each possess one of five different accessory domains, which have roles in ubiquitination, origin of DNA replication recognition or single-stranded nucleic acid binding activity. Using a robust phylogenetic approach we examined each Pif1 class, which revealed that gene duplication, fusion and horizontal gene transfer events have all contributed to the evolution of these enzymes. This study has identified the first collection of Pif1 helicases to contain additional domains, which likely confer novel enzymatic activity, or improve existing functionality. Furthermore, the potential functions of these helicases may shed further light on the overall role the Pif1 family plays in genome maintenance.

Structural Studies of Pif1 Helicases from Thermophilic Bacteria.

Pif1 proteins are DNA helicases belonging to Superfamily 1, with 5' to 3' directionality. They are conserved from bacteria to human and have been shown to be particularly important in eukaryotes for replication and nuclear and mitochondrial genome stability. However, Pif1 functions in bacteria are less known. While most Pif1 from mesophilic bacteria consist of the helicase core with limited N-terminal and C-terminal extensions, some Pif1 from thermophilic bacteria exhibit a C-terminal WYL domain. We solved the crystal structures of Pif1 helicase cores from thermophilic bacteria Deferribacter desulfuricans and Sulfurihydrogenibium sp. in apo and nucleotide bound form. We show that the N-terminal part is important for ligand binding. The full-length Pif1 helicase was predicted based on the Alphafold algorithm and the nucleic acid binding on the Pif1 helicase core and the WYL domain was modelled based on known crystallographic structures. The model predicts that amino acids in the domains 1A, WYL, and linker between the Helicase core and WYL are important for nucleic acid binding. Therefore, the N-terminal and C-terminal extensions may be necessary to strengthen the binding of nucleic acid on these Pif1 helicases. This may be an adaptation to thermophilic conditions.

Quadruplex-Forming Motif Inserted into 3'UTR of Ty1his3-AI Retrotransposon Inhibits Retrotransposition in Yeast.

Guanine quadruplexes (G4s) serve as regulators of replication, recombination and gene expression. G4 motifs have been recently identified in LTR retrotransposons, but their role in the retrotransposon life-cycle is yet to be understood. Therefore, we inserted G4s into the 3'UTR of Ty1his3-AI retrotransposon and measured the frequency of retrotransposition in yeast strains BY4741, Y00509 (without Pif1 helicase) and with G4-stabilization by N-methyl mesoporphyrin IX (NMM) treatment. We evaluated the impact of G4s on mRNA levels by RT-qPCR and products of reverse transcription by Southern blot analysis. We found that the presence of G4 inhibited Ty1his3-AI retrotransposition. The effect was stronger when G4s were on a transcription template strand which leads to reverse transcription interruption. Both NMM and Pif1p deficiency reduced the retrotransposition irrespective of the presence of a G4 motif in the Ty1his3-AI element. Quantity of mRNA and products of reverse transcription did not fully explain the impact of G4s on Ty1his3-AI retrotransposition indicating that G4s probably affect some other steps of the retrotransposon life-cycle (e.g., translation, VLP formation, integration). Our results suggest that G4 DNA conformation can tune the activity of mobile genetic elements that in turn contribute to shaping the eukaryotic genomes.

Inactivation of Pif1 helicase causes a mitochondrial myopathy in mice.

Mutations in genes coding for mitochondrial helicases such as TWINKLE and DNA2 are involved in mitochondrial myopathies with mtDNA instability in both human and mouse. We show that inactivation of Pif1, a third member of the mitochondrial helicase family, causes a similar phenotype in mouse. pif1-/- animals develop a mitochondrial myopathy with respiratory chain deficiency. Pif1 inactivation is responsible for a deficiency to repair oxidative stress-induced mtDNA damage in mouse embryonic fibroblasts that is improved by complementation with mitochondrial isoform mPif1(67). These results open new perspectives for the exploration of patients with mtDNA instability disorders.

Structural and Functional Insights into the Unwinding Mechanism of Bacteroides sp Pif1.

Pif1 is a conserved SF1B DNA helicase involved in maintaining genome stability through unwinding double-stranded DNAs (dsDNAs), DNA/RNA hybrids, and G quadruplex (G4) structures. Here, we report the structures of the helicase domain of human Pif1 and Bacteroides sp Pif1 (BaPif1) in complex with ADP-AlF4(-) and two different single-stranded DNAs (ssDNAs). The wedge region equivalent to the ß hairpin in other SF1B DNA helicases folds into an extended loop followed by an α helix. The Pif1 signature motif of BaPif1 interacts with the wedge region and a short helix in order to stabilize these ssDNA binding elements, therefore indirectly exerting its functional role. Domain 2B of BaPif1 undergoes a large conformational change upon concomitant binding of ATP and ssDNA, which is critical for Pif1's activities. BaPif1 cocrystallized with a tailed dsDNA and ADP-AlF4(-), resulting in a bound ssDNA bent nearly 90° at the ssDNA/dsDNA junction. The conformational snapshots of BaPif1 provide insights into the mechanism governing the helicase activity of Pif1.

Determination of the biochemical properties of full-length human PIF1 ATPase.

The PIF1 helicase family performs many cellular functions. To better understand the functions of the human PIF1 helicase, we characterized the biochemical properties of its ATPase. PIF1 is very sensitive to temperature, whereas it is not affected by pH, and the ATPase activity of human PIF1 is dependent on the divalent cations Mg (2+) and Mn (2+) but not Ca (2+) and Zn (2+). Inhibition was observed when single-stranded DNA was coated with RPA or SSB. Moreover, the ATPase activity of PIF1 proportionally decreased with decreasing oligonucleotide length due to a decreased binding ability. A minimum of 10 oligonucleotide bases are required for PIF1 binding and the hydrolysis of ATP. The analysis of the biochemical properties of PIF1 together with numerous genetic observations should aid in the understanding of its cellular functions.

Roles of PIF1 helicase in cell cycle arrest induced by ionizing radiation / 军事医学

Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.

The purification and preliminary functional study of N-terminal truncated human PIF1 helicases / 西安交通大学学报(医学版)

Objective To elucidate physiological functions of human PIF1 helicase at the molecular level,purify N-terminal truncated PIF1 helicase,PIF1△N,and assay its biochemical properties.Methods The N-terminal cDNA sequence of PIF1 helicase was amplified by PCR using the Hela cell cDNA library as template.The cDNA with a histidine tag at the N-terminus was inserted into the pET20b vector to produce recombinant plasmid.The recombinant PIF1△N was successfully expressed by co-transforming a plasmid encoding rare rRNA.At 4 ℃ through a series of affinity column the recombinant PIF1△N protein was purified by fast protein liquid chromatograph.The biochemical activity of PIF1△N was assayed.Results The cDNA fragment of human PIF1 from 540～1 923 was cloned from Hela cDNA library,and the recombinant PIF1△N protein was successfully overexpressed in E.coli.The purification procedure of PIF1△N protein was established and its biochemical activity was identified.Conclusion N-terminal truncated PIF1 helicase,PIF1△N,has ATPase activity,which is DNA and Mg2+ dependent.