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Molecular and Cellular Biology, April 2006, p. 2490-2500, Vol. 26, No. 7
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.7.2490-2500.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evidence Suggesting that Pif1 Helicase Functions in DNA Replication with the Dna2 Helicase/Nuclease and DNA Polymerase {delta}

Martin E. Budd,1 Clara C. Reis,1,3 Stephanie Smith,2 Kyungjae Myung,2 and Judith L. Campbell1*

Braun Laboratories, California Institute of Technology, Pasadena, California 91125,1 Genetics & Molecular Biology Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892-4442,2 Gulbenkian Ph.D. Program in Biomedicine, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal3

Received 3 December 2005/ Returned for modification 2 January 2006/ Accepted 12 January 2006

The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1{Delta} dna2{Delta} strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol {delta}, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32{Delta} strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol {delta} and/or by an as yet unidentified helicase. Thus, suppression of dna2{Delta} can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1{Delta} mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.

* Corresponding author. Mailing address: Braun Laboratories, 147-75, California Institute of Technology, Pasadena, CA 91125. Phone: (626) 395-6053. Fax: (626) 449-0756. E-mail: jcampbel{at}caltech.edu.

Molecular and Cellular Biology, April 2006, p. 2490-2500, Vol. 26, No. 7
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.7.2490-2500.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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