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J. Cell Biol. 158 (5): 863-872

Copyright © 2002 by the Rockefeller University Press.


Article

DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

Matthew P. Stokes1, Ruth Van Hatten1, Howard D. Lindsay2, and W. Matthew Michael1

1 The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
2 Genome Damage and Stability Center, University of Sussex Falmer, Brighton BN1 9QG, UK

Address correspondence to W. Matthew Michael, The Biological Laboratories, Dept. of Molecular and Cellular Biology, 16 Divinity Ave., Harvard University, Cambridge, MA 02138. Tel.: (617) 496-2940. Fax: (617) 384-7423. E-mail: matt{at}mcb.harvard.edu

Abstract: Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

Key Words: cell cycle; Rad17; DNA damage; DNA replication; S phase


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