Originally published as Genetics Published Articles Ahead of Print on August 24, 2009.

Genetics, Vol. 183, 779-791, November 2009, Copyright © 2009
doi:10.1534/genetics.109.107631

Telomerase Is Essential to Alleviate Pif1-Induced Replication Stress at Telomeres

Michael Chang^*,,,1, Brian Luke^,, Claudine Kraft, Zhijian Li^**, Matthias Peter, Joachim Lingner^, and Rodney Rothstein^*

^* Department of Genetics and Development, Columbia University Medical Center, New York, New York 10032, Swiss Institute for Experimental Cancer Research, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland, National Centre of Competence in Research "Frontiers in Genetics," CH-1211 Geneva, Switzerland, Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland and ^** Banting and Best Department of Medical Research and Department of Molecular Genetics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada

¹ Corresponding author: 701 W. 168th St., New York, NY 10032.
E-mail: mc3096{at}columbia.edu

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 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.

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