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Molecular and Cellular Biology, July 2002, p. 4477-4490, Vol. 22, No. 13
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.13.4477-4490.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Conserved Domain of Schizosaccharomyces pombe dfp1+ Is Uniquely Required for Chromosome Stability following Alkylation Damage during S Phase

Amy D. Fung, Jiongwen Ou, Stephanie Bueler, and Grant W. Brown*

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Received 18 December 2001/ Returned for modification 31 January 2002/ Accepted 25 March 2002

The fission yeast Dbf4 homologue Dfp1 has a well-characterized role in regulating the initiation of DNA replication. Sequence analysis of Dfp1 homologues reveals three highly conserved regions, referred to as motifs N, M, and C. To determine the roles of these conserved regions in Dfp1 function, we have generated dfp1 alleles with mutations in these regions. Mutations in motif N render cells sensitive to a broad range of DNA-damaging agents and replication inhibitors, yet these mutant proteins are efficient activators of Hsk1 kinase in vitro. In contrast, mutations in motif C confer sensitivity to the alkylating agent methyl methanesulfonate (MMS) but, surprisingly, not to UV, ionizing radiation, or hydroxyurea. Motif C mutants are poor activators of Hsk1 in vitro but can fulfill the essential function(s) of Dfp1 in vivo. Strains carrying dfp1 motif C mutants have an intact mitotic and intra-S-phase checkpoint, and epistasis analysis indicates that dfp1 motif C mutants function outside of the known MMS damage repair pathways, suggesting that the observed MMS sensitivity is due to defects in recovery from DNA damage. The motif C mutants are most sensitive to MMS during S phase and are partially suppressed by deletion of the S-phase checkpoint kinase cds1. Following treatment with MMS, dfp1 motif C mutants exhibit nuclear fragmentation, chromosome instability, precocious recombination, and persistent checkpoint activation. We propose that Dfp1 plays at least two genetically separable roles in the DNA damage response in addition to its well-characterized role in the initiation of DNA replication and that motif C plays a critical role in the response to alkylation damage, perhaps by restarting or stabilizing stalled replication forks.

* Corresponding author. Mailing address: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Phone: (416) 946-5733. Fax: (416) 978-8548. E-mail: grant.brown{at}utoronto.ca.

Molecular and Cellular Biology, July 2002, p. 4477-4490, Vol. 22, No. 13
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.13.4477-4490.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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