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J. Biol. Chem., Vol. 280, Issue 13, 12413-12421, April 1, 2005

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Activation of Budding Yeast Replication Origins and Suppression of Lethal DNA Damage Effects on Origin Function by Ectopic Expression of the Co-chaperone Protein Mge1^*

Peter A. Trabold, Martin Weinberger, Li Feng, and William C. Burhans

From the Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York 14263

Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.

Received for publication, October 4, 2004 , and in revised form, January 7, 2005.

^* This research was supported by Public Health Service Grants CA84086 and CA81326 and by shared resources funded by the Roswell Park Cancer Center Support Grant P30 CA16056. 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.

Present address: Dept. of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093.

To whom correspondence should be addressed. Tel.: 716-845-7691; Fax: 716-845-5908; E-mail: wburhans{at}acsu.buffalo.edu.

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