Dynamic Regulation of Single-Stranded Telomeres in Saccharomyces cerevisiae

Genome Instability Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Genetics (Impact Factor: 5.96). 03/2008; 178(2):693-701. DOI: 10.1534/genetics.107.081091
Source: PubMed


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.

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    • "Our finding for the preferential removing of telomerase from long ssDNA by Pif1 could have several implications. First, the ssDNA gap size dependence of telomerase removal by Pif1 helicases seen here could potentially support the previous finding that Pif1 deletion recovered the temperature sensitivity and the increased overall telomere length in the yeast ku mutant cells (41). Yeast ku mutants have been shown to have long telomere overhangs and short telomeres. "
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    ABSTRACT: Telomerase, a ribonucleoprotein complex, is responsible for maintaining the telomere length at chromosome ends. Using its RNA component as a template, telomerase uses its reverse transcriptase activity to extend the 3′-end single-stranded, repetitive telomeric DNA sequence. Pif1, a 5′-to-3′ helicase, has been suggested to regulate telomerase activity. We used single-molecule experiments to directly show that Pif1 helicase regulates telomerase activity by removing telomerase from telomere ends, allowing the cycling of the telomerase for additional extension processes. This telomerase removal efficiency increases at longer ssDNA gaps and at higher Pif1 concentrations. The enhanced telomerase removal efficiency by Pif1 at the longer single-stranded telomeric DNA suggests a way of how Pif1 regulates telomerase activity and maintains telomere length.
    Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku541 · 9.11 Impact Factor
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    • "One complicating factor in characterizing ogg1D strain phenotype is the vicinity of the PIF1 gene. As Pif1p has been shown to be a negative regulator of telomere length (Schulz and Zakian, 1994; Boule and Zakian, 2007; Vega et al, 2007; Smith et al, 2008), Askree et al (2004) have suggested that the telomere-lengthening phenotype observed in ogg1D strain could be caused by altered PIF1 expression or function. We thus checked whether the expression of PIF1 was altered in ogg1D strain. "
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    ABSTRACT: Telomeres consist of short guanine-rich repeats. Guanine can be oxidized to 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG). 8-oxoguanine DNA glycosylase (Ogg1) repairs these oxidative guanine lesions through the base excision repair (BER) pathway. Here we show that in Saccharomyces cerevisiae ablation of Ogg1p leads to an increase in oxidized guanine level in telomeric DNA. The ogg1 deletion (ogg1Delta) strain shows telomere lengthening that is dependent on telomerase and/or Rad52p-mediated homologous recombination. 8-oxoG in telomeric repeats attenuates the binding of the telomere binding protein, Rap1p, to telomeric DNA in vitro. Moreover, the amount of telomere-bound Rap1p and Rif2p is reduced in ogg1Delta strain. These results suggest that oxidized guanines may perturb telomere length equilibrium by attenuating telomere protein complex to function in telomeres, which in turn impedes their regulation of pathways engaged in telomere length maintenance. We propose that Ogg1p is critical in maintaining telomere length homoeostasis through telomere guanine damage repair, and that interfering with telomere length homoeostasis may be one of the mechanism(s) by which oxidative DNA damage inflicts the genome.
    The EMBO Journal 11/2009; 29(2):398-409. DOI:10.1038/emboj.2009.355 · 10.43 Impact Factor
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    • "In S. cerevisiae, overexpression of PIF1 reduces the viability of yku70, yku80, and cdc13 mutants (Banerjee et al. 2006; Vega et al. 2007) while absence of Pif1 rescues the temperature sensitivity of these mutants (Downey et al. 2006; Vega et al. 2007; Smith et al. 2008). Yku70 and Yku80 form the heterodimeric Ku complex that protects telomeres from degradation and inappropriate recombination (Gravel et al. 1998; Polotnianka et al. 1998; Dubois et al. 2002; Maringele and Lydall 2002). "
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    ABSTRACT: Pif1, an evolutionarily conserved helicase, negatively regulates telomere length by removing telomerase from chromosome ends. Pif1 has also been implicated in DNA replication processes such as Okazaki fragment maturation and replication fork pausing. We find that overexpression of Saccharomyces cervisiae PIF1 results in dose-dependent growth inhibition. Strong overexpression causes relocalization of the DNA damage response factors Rfa1 and Mre11 into nuclear foci and activation of the Rad53 DNA damage checkpoint kinase, indicating that the toxicity is caused by accumulation of DNA damage. We screened the complete set of approximately 4800 haploid gene deletion mutants and found that moderate overexpression of PIF1, which is only mildly toxic on its own, causes growth defects in strains with mutations in genes involved in DNA replication and the DNA damage response. Interestingly, we find that telomerase-deficient strains are also sensitive to PIF1 overexpression. Our data are consistent with a model whereby increased levels of Pif1 interfere with DNA replication, causing collapsed replication forks. At chromosome ends, collapsed forks result in truncated telomeres that must be rapidly elongated by telomerase to maintain viability.
    Genetics 09/2009; 183(3):779-91. DOI:10.1534/genetics.109.107631 · 5.96 Impact Factor
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