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1.
The Pif1 family of DNA helicases is conserved from yeast to humans. Although the helicase domains of family members are well conserved, the amino termini of these proteins are not. The Saccharomyces cerevisiae genome encodes two Pif1 family members, Rrm3p and Pif1p, that have very different functions. To determine if the amino terminus of Rrm3p contributes to its role in promoting fork progression at >1000 discrete chromosomal sites, we constructed a deletion series that lacked portions of the 249-amino-acid amino terminus. The phenotypes of cells expressing alleles that lacked all or most of the amino terminus were indistinguishable from those of rrm3Delta cells. Rrm3p deletion derivatives that lacked smaller portions of the amino terminus were also defective, but the extent of replication pausing at tRNA genes, telomeres, and ribosomal DNA (rDNA) was not as great as in rrm3Delta cells. Deleting only 62 amino acids from the middle of the amino terminus affected only rDNA replication, suggesting that the amino terminus can confer locus-specific effects. Cells expressing a fusion protein consisting of the Rrm3p amino terminus and the Pif1p helicase domain displayed defects similar to rrm3Delta cells. These data demonstrate that the amino terminus of Rrm3p is essential for Rrm3p function. However, the helicase domain of Rrm3p also contributes to its functional specificity. 相似文献
2.
Schizosaccharomyces pombe pfh1+ encodes an essential 5' to 3' DNA helicase that is a member of the PIF1 subfamily of DNA helicases
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Zhou JQ Qi H Schulz VP Mateyak MK Monson EK Zakian VA 《Molecular biology of the cell》2002,13(6):2180-2191
The Saccharomyces cerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans. S. cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA. Here we describe the isolation and characterization of pfh1+, a Schizosaccharomyces pombe gene that encodes a Pif1-like protein. Pfh1p was the only S. pombe protein with high identity to Saccharomyces Pif1p. Unlike the two S. cerevisiae Pif1 subfamily proteins, the S. pombe Pfh1p was essential. Like Saccharomyces Pif1p, a truncated form of the S. pombe protein had 5' to 3' DNA helicase activity. Point mutations in an invariant lysine residue in the ATP binding pocket of Pfh1p had the same phenotype as deleting pfh1+, demonstrating that the ATPase/helicase activity of Pfh1p was essential. Although mutant spores depleted for Pfh1p proceeded through S phase, they arrested with a terminal cellular phenotype consistent with a postinitiation defect in DNA replication. Telomeric DNA was modestly shortened in the absence of Pfh1p. However, genetic analysis demonstrated that maintenance of telomeric DNA was not the sole essential function of S. pombe Pfh1p. 相似文献
3.
Vega LR Phillips JA Thornton BR Benanti JA Onigbanjo MT Toczyski DP Zakian VA 《PLoS genetics》2007,3(6):e105
The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation. 相似文献
4.
The Pif1p family of DNA helicases is conserved from yeast to humans. To date, four members of this family have been analyzed in some detail by in vitro and in vivo assays: the two baker's yeast helicases, ScPif1p and Rrm3p, the fission yeast Pfh1p and the human enzyme hPif1p. In vitro, these enzymes are 5′ to 3′ DNA helicase and show little processivity. In vivo, ScPif1p, Rrm3p and probably Pfh1p, function in both the nucleus at specific genomic loci and in mitochondria, where they are needed for the stable maintenance of the genome as accessory helicases to the replication machinery. Interestingly, they act on common DNA substrates but appear to have largely non-overlapping cellular functions, ranging from Okazaki fragment processing, telomerase inhibition, to helping the replication fork progress through non-nucleosomal protein–DNA complexes. For example, both ScPif1p and Rrm3p affect the replication of telomeres, but in a different way: Pif1p inhibits telomerase-mediated telomere elongation by directly removing telomerase from a DNA end, whereas Rrm3p facilitates replication through telomeric DNA. Here we review the current knowledge on the Pif1-like helicases, as a first step towards understanding the basis of their functional specialization and mechanism of action. 相似文献
5.
Saccharomyces cerevisiae Piflp helicase is the founding member of the Pifl subfamily that isconserved from yeast to human.The potential human homolog of the yeast PIFI gene has been cloned fromthe cDNA library of the Hek293 cell line.Here,we described a purification procedure of glutathione S-transferase(GST)-fused N terminal truncated human Pifl protein(hPif1ΔN)from yeast and characterizedthe enzymatic kinetics of its ATP hydrolysis activity.The ATPase activity of human Pif1 is dependent ondivalent cation,such as Mg~(2 ),Ca~(2 )and single-stranded DNA.Km for ATP for the ATPase activity isapproximately 200 μM.As the ATPase activity is essential for hPifl's helicase activity,these results willfacilitate the further investigation on hPif1. 相似文献
6.
The Schizosaccharomyces pombe Pfh1p DNA helicase is essential for the maintenance of nuclear and mitochondrial DNA 总被引:2,自引:0,他引:2
Schizosaccharomyces pombe Pfh1p is an essential member of the Pif family of 5′-3′ DNA helicases. The two Saccharomyces cerevisiae homologs, Pif1p and Rrm3p, function in nuclear DNA replication, telomere length regulation, and mitochondrial genome integrity. We demonstrate here the existence of multiple Pfh1p isoforms that localized to either nuclei or mitochondria. The catalytic activity of Pfh1p was essential in both cellular compartments. The absence of nuclear Pfh1p resulted in G2 arrest and accumulation of DNA damage foci, a finding suggestive of an essential role in DNA replication. Exogenous DNA damage resulted in localization of Pfh1p to DNA damage foci, suggesting that nuclear Pfh1p also functions in DNA repair. The absence of mitochondrial Pfh1p caused rapid depletion of mitochondrial DNA. Despite localization to nuclei and mitochondria in S. pombe, neither of the S. cerevisiae homologs, nor human PIF1, suppressed the lethality of pfh1Δ cells. However, the essential nuclear function of Pfh1p could be supplied by Rrm3p. Expression of Rrm3p suppressed the accumulation of DNA damage foci but not the hydroxyurea sensitivity of cells depleted of nuclear Pfh1p. Together, these data demonstrate that Pfh1p has essential roles in the replication of both nuclear and mitochondrial DNA. 相似文献
7.
Woo-Hyun Chung 《Journal of microbiology (Seoul, Korea)》2014,52(2):89-98
Pif1 DNA helicase is the prototypical member of a 5′ to 3′ helicase superfamily conserved from bacteria to humans. In Saccharomyces cerevisiae, Pif1 and its homologue Rrm3, localize in both mitochondria and nucleus playing multiple roles in the maintenance of genomic homeostasis. They display relatively weak processivities in vitro, but have largely non-overlapping functions on common genomic loci such as mitochondrial DNA, telomeric ends, and many replication forks especially at hard-to-replicate regions including ribosomal DNA and G-quadruplex structures. Recently, emerging evidence shows that Pif1, but not Rrm3, has a significant new role in repair-associated DNA synthesis with Polδ during homologous recombination stimulating D-loop migration for conservative DNA replication. Comparative genetic and biochemical studies on the structure and function of Pif1 family helicases across different biological systems are further needed to elucidate both diversity and specificity of their mechanisms of action that contribute to genome stability. 相似文献
8.
The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replication fork progression in ribosomal DNA 总被引:4,自引:0,他引:4
Replication of Saccharomyces ribosomal DNA (rDNA) proceeds bidirectionally from origins in a subset of the approximately 150 tandem repeats, but the leftward-moving fork stops when it encounters the replication fork barrier (RFB). The Pif1p helicase and the highly related Rrm3p were rDNA associated in vivo. Both proteins affected rDNA replication but had opposing effects on fork progression. Pif1p helped maintain the RFB. Rrm3p appears to be the replicative helicase for rDNA as it acted catalytically to promote fork progression throughout the rDNA. Loss of Rrm3p increased rDNA breakage and accumulation of rDNA circles, whereas breakage and circles were less common in pif1 cells. These data support a model in which replication fork pausing causes breakage and recombination in the rDNA. 相似文献
9.
Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres. 相似文献
10.
Def1p is involved in telomere maintenance in budding yeast 总被引:3,自引:0,他引:3
Saccharomyces Rrm3p, a member of Pif1 5'-3' DNA helicase subfamily, helps replication forks traverse protein-DNA complexes, including the telomere. Here we have identified an Rrm3p interaction protein known to be Def1p. In def1 mutants, telomeres were approximately 200-bp shorter than that in wild-type cells. DEF1 is also required for the stable maintenance of mitochondrial DNA, and the telomere shortening phenotype seen in def1 cells is not a secondary consequence of the mitochondrion defect. A combination of DEF1 null mutation with deletion of EST2 or EST3 resulted in an accelerated senescence phenotype, suggesting that Def1p is not involved in the telomerase recruitment pathway. In the absence of telomerase, cells escape senescence by either amplifying Y' regions or TG-telomeric repeats to generate type I or type II survivors, respectively. Only type I survivors were recovered from both def1Delta est2Delta and def1Delta est3Delta double mutant cells, further suggesting that the function of Def1p in telomere maintenance is specific. Our novel findings of the functions of Def1p in telomere and mitochondria suggested that Def1p plays multiple roles in yeast. 相似文献
11.
How the cellular amount of mitochondrial DNA (mtDNA) is regulated under normal conditions and in the presence of genotoxic
stress is less understood. We demonstrate that the inefficient mtDNA replication process of mutant yeast cells lacking the
PIF1 DNA helicase is partly rescued in the absence of the DNA helicase RRM3. The rescue effect is likely due to the increase in the deoxynucleoside triphosphates (dNTPs) pool caused by the lack of
RRM3. In contrast, the Pif1p-dependent mtDNA breakage in the presence and absence of genotoxic stress is not suppressed if RRM3 is lacking suggesting that this phenotype is likely independent of the dNTP pool. Pif1 protein (Pif1p) was found to stimulate
the incorporation of dNTPs into newly synthesised mtDNA of gradient-purified mitochondria. We propose that Pif1p that acts
likely as a DNA helicase in mitochondria affects mtDNA replication directly. Possible roles of Pif1p include the resolution
of secondary DNA and/or DNA/RNA structures, the temporarily displacement of tightly bound mtDNA-binding proteins, or the stabilization
of the mitochondrial replication complex during mtDNA replication.
X. Cheng, Y. Qin contributed equally to this work. 相似文献
12.
人类重组PIF1蛋白的表达纯化和解螺旋酶活性的分析 总被引:1,自引:0,他引:1
Pif1解螺旋酶家族在酵母到人的进化中非常保守, 在生物体内具有很多重要的生理作用。为了从生物化学水平研究人类PIF1的功能, 从HeLa细胞的cDNA文库中克隆得到人类PIF1全长基因, 通过共转化一个携带稀有遗传密码tRNA1的质粒和一个编码分子伴侣的质粒, 增加了PIF1蛋白在大肠杆菌中的表达, 最后通过快速液相色谱纯化系统, 采用亲和层析和凝胶过滤, 纯化了人类重组PIF1蛋白。生物化学活性检测证明了纯化的人类PIF1蛋白具有ATP酶及解螺旋酶活性。人类PIF1蛋白的纯化为我们从分子水平理解PIF1在体内的功能奠定了基础。 相似文献
13.
Tanaka H Ryu GH Seo YS Tanaka K Okayama H MacNeill SA Yuasa Y 《Nucleic acids research》2002,30(21):4728-4739
The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease–helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1+, encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5′ to 3′ DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the comple tion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage. 相似文献
14.
Pif1p is the prototypical member of the PIF1 family of DNA helicases, a subfamily of SFI helicases conserved from yeast to humans. Baker's yeast Pif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and may also have a general role in chromosomal replication by affecting Okazaki fragment maturation. Here we investigate the substrate preferences for Pif1p. The enzyme was preferentially active on RNA–DNA hybrids, as seen by faster unwinding rates on RNA–DNA hybrids compared to DNA–DNA hybrids. When using forked substrates, which have been shown previously to stimulate the enzyme, Pif1p demonstrated a preference for RNA–DNA hybrids. This preferential unwinding could not be correlated to preferential binding of Pif1p to the substrates that were the most readily unwound. Although the addition of the single-strand DNA-binding protein replication protein A (RPA) stimulated the helicase reaction on all substrates, it did not diminish the preference of Pif1p for RNA–DNA substrates. Thus, forked RNA–DNA substrates are the favored substrates for Pif1p in vitro. We discuss these findings in terms of the known biological roles of the enzyme. 相似文献
15.
16.
Requirement of Rrm3 helicase for repair of spontaneous DNA lesions in cells lacking Srs2 or Sgs1 helicase
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The Rrm3 DNA helicase of Saccharomyces cerevisiae interacts with proliferating cell nuclear antigen and is required for replication fork progression through ribosomal DNA repeats and subtelomeric and telomeric DNA. Here, we show that rrm3 srs2 and rrm3 sgs1 mutants, in which two different DNA helicases have been inactivated, exhibit a severe growth defect and undergo frequent cell death. Cells lacking Rrm3 and Srs2 arrest in the G(2)/M phase of the cell cycle with 2N DNA content and frequently contain only a single nucleus. The phenotypes of rrm3 srs2 and rrm3 sgs1 mutants were suppressed by disrupting early steps of homologous recombination. These observations identify Rrm3 as a new member of a network of pathways, involving Sgs1 and Srs2 helicases and Mus81 endonuclease, suggested to act during repair of stalled replication forks. 相似文献
17.
The temperature-sensitive phenotypes of yku70Delta and yku80Delta have provided a useful tool for understanding telomere homeostasis. Mutating the helicase domain of the telomerase inhibitor Pif1 resulted in the inactivation of cell cycle checkpoints and the subsequent rescue of temperature sensitivity of the yku70Delta strain. The inactivation of Pif1 in yku70Delta increased overall telomere length. However, the long G-rich, single-stranded overhangs at the telomeres, which are the major cause of temperature sensitivity, were slightly increased. Interestingly, the rescue of temperature sensitivity in strains having both pif1-m2 and yku70Delta mutations depended on the homologous recombination pathway. Furthermore, the BLM/WRN helicase yeast homolog Sgs1 exacerbated the temperature sensitivity of the yku70Delta strain. Therefore, the yKu70-80 heterodimer and telomerase maintain telomere size, and the helicase activity of Pif1 likely also helps to balance the overall size of telomeres and G-rich, single-stranded overhangs in wild-type cells by regulating telomere protein homeostasis. However, the absence of yKu70 may provide other proteins such as those involved in homologous recombination, Sgs1, or Pif1 additional access to G-rich, single-stranded DNA and may determine telomere size, cell cycle checkpoint activation, and, ultimately, temperature sensitivity. 相似文献
18.
Mitochondrial DNA (mtDNA) is highly susceptible to oxidative and chemically induced damage, and these insults lead to a number of diseases. In Saccharomyces cerevisiae, the DNA helicase Pif1p is localized to the nucleus and mitochondria. We show that pif1 mutant cells are sensitive to ethidium bromide-induced damage and this mtDNA is prone to fragmentation. We also show that Pif1p associates with mtDNA. In pif1 mutant cells, mtDNA breaks at specific sites that exhibit Pif1-dependent recombination. We conclude that Pif1p participates in the protection from double-stranded (ds) DNA breaks or alternatively in the repair process of dsDNA breaks in mtDNA. 相似文献
19.
Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins. 相似文献
20.
Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing. 相似文献