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J. Biol. Chem., Vol. 279, Issue 51, 53023-53027, December 17, 2004

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DNA Binding Domain in the Replication Checkpoint Protein Mrc1 of Schizosaccharomyces pombe^*

Hui Zhao and Paul Russell¶||

From the Departments of Molecular Biology and ^¶Cell Biology, The Scripps Research Institute, La Jolla, California 92037

The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides. This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis. In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1. In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45. Whether or not Mrc1 interacts directly with DNA is unknown. Here we define a 150 amino acid DNA binding domain (DBD) in the N-terminal region of S. pombe Mrc1. The DBD interacts preferentially with branched DNA structures in vitro. Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea. These mutations also impair the replication checkpoint arrest. The DBD has a helix-loop-helix motif that is predicted to bind DNA. This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins.

Received for publication, September 1, 2004

^* This work was supported by National Institutes of Health Grant GM59477 (to P. R.). 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 Biology, Pfizer, San Diego, CA 92121.

^|| To whom correspondence should be addressed. Tel.: 858-784-8273; Fax: 858-784-2265; E-mail: prussell{at}scripps.edu.

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