Brief CommunicationThe Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening
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)
- et al.
Telomere maintenance in fission yeast requires an Est1 ortholog
Curr. Biol.
(2003) - et al.
Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication
Cell
(2009) - et al.
Mammalian telomeres end in a large duplex loop
Cell
(1999) - et al.
Taz1 binding to a fission yeast model telomere: formation of telomeric loops and higher order structures
J. Biol. Chem.
(2004) - et al.
Unwinding the functions of the Pif1 family helicases
DNA Repair
(2010) - et al.
The saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation
Cell
(1994) - et al.
Sumoylation of RecQ helicase controls the fate of dysfunctional telomeres
Mol. Cell
(2009) - et al.
The localization of replication origins on ARS plasmids in S. cerevisiae
Cell
(1987) - et al.
Protection and replication of telomeres in fission yeast
Biochem. Cell Biol.
(2009) - et al.
Fission yeast Pot1-Tpp1 protects telomeres and regulates telomere length
Science
(2008)
Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA
Nat. Struct. Mol. Biol.
TER1, the RNA subunit of fission yeast telomerase
Nat. Struct. Mol. Biol.
Telomerase catalytic subunit homologs from fission yeast and human
Science
Two modes of survival of fission yeast without telomerase
Science
Saccharomyces Rrm3p, a 5′ to 3′ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA
Genes Dev.
Semi-conservative DNA replication through telomeres requires Taz1
Nature
The Pif1 family in prokaryotes: what are our helicases doing in your bacteria?
Mol. Biol. Cell
The Schizosaccharomyces pombe Pfh1p DNA helicase is essential for the maintenance of nuclear and mitochondrial DNA
Mol. Cell. Biol.
Cited by (0)
- 1
All these authors made equal contribution.