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J. Biol. Chem., Vol. 278, Issue 25, 22513-22522, June 20, 2003

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Dna2 Helicase/Nuclease Causes Replicative Fork Stalling and Double-strand Breaks in the Ribosomal DNA of Saccharomyces cerevisiae^*

Tao Weitao , Martin Budd , Laura L. Mays Hoopes  and Judith L. Campbell  ¶

From the Braun Laboratories 147-75, California Institute of Technology, Pasadena, California 91125 and the Department of Biology and Molecular Biology Program, Pomona College, Seaver South Laboratory, Claremont, California 91711

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with rrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

Received for publication, February 14, 2003 , and in revised form, April 8, 2003.

^* This research was supported by National Institutes of Health Grant GM25508 and National Science Foundation Grant MCB9985527 (to J. L. C.) and National Science Foundation Grant RUI MCB113937 and Pomona College Research Committee Funds (to L. L. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 626-395-6053; Fax: 626-405-9452; E-mail: jcampbel{at}cco.caltech.edu.

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