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Molecular and Cellular Biology, August 2004, p. 6891-6899, Vol. 24, No. 16
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.16.6891-6899.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

Xuan Wang,1 Grzegorz Ira,1 José Antonio Tercero,2 Allyson M. Holmes,1,{dagger} John F. X. Diffley,2 and James E. Haber1*

Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110,1 Cancer Research UK, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom2

Received 7 April 2004/ Returned for modification 11 May 2004/ Accepted 18 May 2004

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4{Delta} strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase {alpha} (Pol{alpha}), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol{alpha} nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol{alpha}-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

* Corresponding author. Mailing address: Rosenstiel Center and Department of Biology, Brandeis University, Waltham, MA 02454-9110. Phone: (781) 736-2462. Fax: (781) 736-2405. E-mail: haber{at}brandeis.edu.

{dagger} Present address: Unité de Dynamique du Génome, URA 1644 du CNRS, Departement de la Structure et Dynamique des Génomes, Institut Pasteur, 75724 Paris Cedex 15, France.

Molecular and Cellular Biology, August 2004, p. 6891-6899, Vol. 24, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.16.6891-6899.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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