Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

George, Tresa; Qin, Wen; Griffiths, Richard; Ganesh, Anil; Meuth, Mark; Sanders, Cyril M.

doi:10.1093/nar/gkp671

Cited by 54 publications

(83 citation statements)

References 40 publications

(54 reference statements)

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“…Thus, it was proposed that PIF1 function is redundant in mammalian cells. Nevertheless, in vitro analysis of purified full-length PIF1 suggested that it bound and unwound DNA structures that might form during DNA replication stress, including structures resembling stalled replication forks (14) or G-quadruplexes (28). The effects of PIF1 depletion on S-phase transition, checkpoint activation, and apoptosis in tumor cells are consistent with these in vitro results; however, nontumor cell lines did not show similar responses.…”

Section: Discussionsupporting

confidence: 61%

“…In vitro assays of PIF1 binding and unwinding on artificial substrates resembling replication forks (14) suggested that PIF1 could play a role in the generation of ssDNA that triggers activation of CHK1. Consistent with this argument, CHK1 activation, which is dependent upon ssDNA formation (23), was lower in PIF1-depleted cells exposed to thymidine.…”

Section: Discussionmentioning

confidence: 99%

“…However in vitro studies of purified PIF1 protein indicate that it can bind and unwind DNA structures resembling stalled replication forks to produce free 3 0 ssDNA ends, suggesting that PIF1 could play a role in the response to DNA replication stress (14). The aim of the work reported here was to investigate the potential role of PIF1 in the cellular response to DNA replication stress.…”

Section: Introductionmentioning

confidence: 99%

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Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

et al. 2011

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Defining the processes that sustain telomere maintenance is critical to our understanding of cancer and longevity. PIF1 is a nonprocessive 5 0 ->3 0 human DNA helicase that exhibits broad substrate specificity. In vitro studies have implicated PIF1 in maintaining telomeres and processing stalled DNA replication forks, but disruption of the murine Pif1 gene did not yield any apparent phenotype. In this study, we evaluated the function of the PIF1 gene in human cells by using siRNA knockdown strategies to gauge its role in the response to DNA replication stress. We found that PIF1 depletion reduced the survival of both p53-deficient and p53-proficient human tumor cells by triggering apoptosis. In contrast, nonmalignant cells were unaffected by PIF1 depletion. Apoptosis induction in tumor cells was augmented by cotreatment with replication inhibitors (thymidine, hydroxyurea, or gemcitabine). When sensitive PIF1-depleted cells were released from a thymidine-induced S-phase arrest, there remained a subpopulation of cells that failed to enter S-phase. This cell subpopulation displayed an increase in levels of cyclin E and p21, as well as a deficiency in S-phase checkpoint markers that were induced with thymidine in PIF1 expressing cells. Specifically, CHK1 activation was suppressed and we detected no consistent changes in ATM S1981 autophosphorylation, gH2AX induction, or RPA hyperphosphorylation. Death in PIF1-depleted cells was detected in late G 1 /early S-phase and was dependent on caspase-3 activity. Taken together, our findings suggest roles for PIF1 in S-phase entry and progression that are essential to protect human tumor cells from apoptosis. Cancer Res; 71(14); 4998-5008. '2011 AACR.

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Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

et al. 2011

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“…It is also possible that Mcm2-7 may be unable to unwind the final portion of DNA between the converging replication forks, leaving only Pfh1 to perform this function. In this regard, it is interesting to note that human Pif1 can unwind synthetic replication forks in vitro in a way that could promote progression of the leading strand polymerase in vivo (George et al 2009). …”

Section: Discussionmentioning

confidence: 99%

The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability

Steinacher¹,

Osman²,

Dalgaard³

et al. 2012

Genes Dev.

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Bidirectionally moving DNA replication forks merge at termination sites composed of accidental or programmed DNA-protein barriers. If merging fails, then regions of unreplicated DNA can result in the breakage of DNA during mitosis, which in turn can give rise to genome instability. Despite its importance, little is known about the mechanisms that promote the final stages of fork merging in eukaryotes. Here we show that the Pif1 family DNA helicase Pfh1 plays a dual role in promoting replication fork termination. First, it facilitates replication past DNA-protein barriers, and second, it promotes the merging of replication forks. A failure of these processes in Pfh1-deficient cells results in aberrant chromosome segregation and heightened genome instability.

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“…ScRrm3 also promotes fork progression at other sites where ScPif1 is not known to act, such as at tRNA genes, inactive origins, and the silent mating type loci, while ScPif1 is important for replication through G-rich motifs that can form G-quadruplex structures in vitro (Ivessa et al 2003;Paeschke et al 2011). Human Pif1 (hPif1) also unwinds G-quadruplex structures in vitro, as well as DNA structures that resemble stalled replication forks (George et al 2009;Sanders 2010). Although S. cerevisiae Pif1 family helicases have important roles in DNA replication, pif1D rrm3D cells are viable (Ivessa et al 2000).…”

mentioning

confidence: 99%

DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase

Sabouri¹,

McDonald²,

Webb³

et al. 2012

Genes Dev.

127

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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.

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Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

Cited by 54 publications

References 40 publications

Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability

DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase

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