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Molecular and Cellular Biology, September 2009, p. 5008-5019, Vol. 29, No. 18
0270-7306/09/$08.00+0     doi:10.1128/MCB.01934-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress{triangledown}

Makiko Komata,1 Masashige Bando,1 Hiroyuki Araki,2 and Katsuhiko Shirahige1*

Laboratory of Chromosome Structure and Function, Department of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-20, 4259, Nagatsuta, Midori-ku, Yokohama City, Kanagawa 226-8501 Japan,1 Division of Microbial Genetics, National Institute of Genetics, Research Organization of Information and Systems, 1111 Yata, Mishima, Shizuoka 411-8540, Japan2

Received 22 December 2008/ Returned for modification 21 January 2009/ Accepted 6 July 2009

Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

* Corresponding author. Mailing address: Laboratory of Chromosome Structure and Function, Department of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-20, 4259, Nagatsuta, Midori-ku, Yokohama City, Kanagawa 226-8501, Japan. Phone: 81-45-924-5812. Fax: 81-45-924-5814. E-mail: kshirahi{at}bio.titech.ac.jp

{triangledown} Published ahead of print on 20 July 2009.

Molecular and Cellular Biology, September 2009, p. 5008-5019, Vol. 29, No. 18
0270-7306/09/$08.00+0     doi:10.1128/MCB.01934-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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