Elsevier

DNA Repair

Volume 24, December 2014, Pages 80-86
DNA Repair

Brief Communication
The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening

https://doi.org/10.1016/j.dnarep.2014.09.008Get rights and content

Highlights

Abstract

Pif1 family helicases are evolutionary conserved 5′–3′ DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.

Introduction

Telomeres, the DNA–protein structures at the ends of eukaryotic chromosomes, are critical for genome stability. Telomeres in the fission yeast Schizosaccharomyces pombe, like their human counterparts, are assembled into a six-membered protein complex called shelterin that protects them from degradation and end-to-end fusions [1]. The S. pombe shelterin consists of Pot1, the sequence specific telomere single-strand binding protein, Taz1, the sequence specific duplex DNA binding protein, Poz1, Ccq1, Rap1, and Tpz1 [1], [2].

Telomeres pose several problems for DNA replication. Conventional DNA polymerases cannot replicate the very ends of linear chromosomes. In virtually all eukaryotes, this problem is solved by telomerase, a telomere dedicated reverse transcriptase that uses its RNA component as a template to lengthen the G-strand of telomeric DNA. The S. pombe telomerase consists minimally of a catalytic subunit Trt1, the templating RNA subunit, TER1 and an accessory subunit, Est1 [3], [4], [5], [6]. Although telomerase is critical for telomere maintenance, in S. pombe, telomerase deficient cells can survive by either chromosome circularization or Rhp51-dependent homologous recombination (ALT, alternative lengthening of telomeres) [7].

Conventional DNA polymerases also have problems during semi-conservative replication of telomeres. In both budding and fission yeast, replication forks move slowly through telomeric DNA positioned at the end or internally on the chromosome, even in wild type cells [8], [9]. In S. pombe and mouse, loss of the duplex telomere binding proteins Taz1 (S. pombe) or TRF1 (mouse) exacerbates problems in telomere replication [9], [10]. In multiple organisms, including humans, chromosomes end in t-loops, which are formed by invasion of the single-stranded G-rich tail of the telomere into duplex telomeric DNA [11]. Although t-loops have not been detected at S. pombe telomeres, incubation of 3′ tailed duplex S. pombe telomeric DNA with Taz1 generates t-loop structures in vitro [12]. T-loops are another challenge to the replication machinery. Taken together, these data suggest that telomeres are hard-to-replicate owing to both their non-nucleosomal protein structure and to the repetitive and G-rich nature of telomeric DNA.

Here we determine if the S. pombe Pfh1 DNA helicase, a member of the Pif1 family of 5′–3′ DNA helicases, affects telomeres [13], [14]. Unlike budding yeast, which encodes two Pif1 helicases, ScPif1 and ScRrm3 (Sc, Saccharomyces cerevisiae), most eukaryotes, including S. pombe and humans encode a single Pif1 family helicase, named, respectively, Pfh1 and hPIF1. The three yeast Pif1 family helicases are multifunctional, with critical roles in maintenance of both nuclear and mitochondrial DNA [14]. In S. pombe, Pfh1 is encoded by an essential gene, and the absence of either the nuclear or the mitochondrial isoform is lethal [15]. Pfh1 facilitates fork progression at many nuclear sites, including highly transcribed RNA polymerase II and III genes, the mating type locus, the rDNA, and converged replication forks [16], [17]. Mutations in hPIF1 are found in families with high risk of breast cancer, and S. pombe cells with the corresponding mutation are not viable [18]. However, the effect of hPIF1 loss on telomere replication is not resolved [19].

So far, all tested eukaryotic Pif1 family helicases function at telomeres. ScPif1 is a negative regulator of telomere length and telomere addition at double-strand breaks that acts by displacing telomerase from DNA ends [20], [21], [22], [23]. Its overexpression results in short telomeres [22], as does overexpression of hPIF1 in human tissue culture cells [24]. In addition, hPIF1 suppresses the long telomere phenotype of pif1 budding yeast cells [25]. Although ScRrm3 does not inhibit telomerase, it promotes fork progression through telomeric DNA [8].

To understand the telomere functions of Pif1 helicases in an organism that expresses only one Pif1 helicase we examined the role of Pfh1 in S. pombe telomere replication. We find that Pfh1 was needed to facilitate fork progression at telomeric repeats, and that this effect is probably direct because telomeres had high Pfh1 association. To resolve conflicting results on the effects of Pfh1 on telomere length, we overexpressed Pfh1, which resulted in telomere lengthening, even in recombination deficient cells, but not in a RPA mutant that has telomerase defects. Thus, Pfh1 is a positive regulator of semi-conservative telomeric DNA replication and performs a unique PIF1 family function in telomerase-mediated telomere lengthening.

Section snippets

Pfh1 facilitates replication fork progression through telomeres

Pfh1 promotes replication through multiple types of hard-to replicate sites [16], [17]. As S. pombe telomeric DNA impedes replication fork progression even in wild type (WT) cells [9], we asked if Pfh1 also affects semi-conservative replication at telomeres. To do so, we examined telomere replication intermediates in a strain (YSA60; Table S1) where Pfh1 was expressed as a GFP fusion under the control of the thiamine-repressible nmt81 promoter (nmt81-pfh1-GFP). The Pfh1-GFP fusion was expressed

Growth conditions, strains and plasmids

All yeast strains used in this study are listed in Table S1. Cells were grown in either yeast extract medium (YES), synthetic minimal medium (EMM) in the presence or absence of 30 μM thiamine or in histidine drop-out EMM media and grown at 30 °C. The mitochondrial only Pfh1 allele, called pfh1-mt* was described previously [15]. Briefly, this allele contains mutations of the methionine codons M265 and M320 to alanine, and M170 to leucine, as well as, the addition of a carboxy-terminal nuclear

Conflict of interests

The authors declare that they have no conflict of interest.

Author contributions

KM, NS, CW, and VZ designed the experiments. KM, NS, and CW performed the experiments. KM, NS, CW, and VZ analyzed the data and wrote the paper.

Acknowledgments

We thank Dr. Matthew Whitby and Dr. Masaru Ueno for strains. NS was supported by Wenner-Gren Foundations and Swedish Society for Medical Research. Work at Princeton was supported by a grant from the US National Institutes of Health (NIH) grant GM43265.

References (37)

  • C.J. Webb et al.

    Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA

    Nat. Struct. Mol. Biol.

    (2008)
  • J. Leonardi et al.

    TER1, the RNA subunit of fission yeast telomerase

    Nat. Struct. Mol. Biol.

    (2008)
  • T.M. Nakamura et al.

    Telomerase catalytic subunit homologs from fission yeast and human

    Science

    (1997)
  • T.M. Nakamura et al.

    Two modes of survival of fission yeast without telomerase

    Science

    (1998)
  • A.S. Ivessa et al.

    Saccharomyces Rrm3p, a 5′ to 3′ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA

    Genes Dev.

    (2002)
  • K.M. Miller et al.

    Semi-conservative DNA replication through telomeres requires Taz1

    Nature

    (2006)
  • M.L. Bochman et al.

    The Pif1 family in prokaryotes: what are our helicases doing in your bacteria?

    Mol. Biol. Cell

    (2011)
  • S.F. Pinter et al.

    The Schizosaccharomyces pombe Pfh1p DNA helicase is essential for the maintenance of nuclear and mitochondrial DNA

    Mol. Cell. Biol.

    (2008)
  • Cited by (0)

    1

    All these authors made equal contribution.

    View full text