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Molecular and Cellular Biology, May 2003, p. 3487-3496, Vol. 23, No. 10
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.10.3487-3496.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Mechanism of Mus81-Mms4 Cleavage Site Selection Distinguishes It from the Homologous Endonuclease Rad1-Rad10

Suzanne A. Bastin-Shanower, William M. Fricke, Janet R. Mullen, and Steven J. Brill*

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854

Received 31 January 2003/ Returned for modification 19 February 2003/ Accepted 21 February 2003

Mus81-Mms4 and Rad1-Rad10 are homologous structure-specific endonucleases that cleave 3' branches from distinct substrates and are required for replication fork stability and nucleotide excision repair, respectively, in the yeast Saccharomyces cerevisiae. We explored the basis of this biochemical and genetic specificity. The Mus81-Mms4 cleavage site, a nick 5 nucleotides (nt) 5' of the flap, is determined not by the branch point, like Rad1-Rad10, but by the 5' end of the DNA strand at the flap junction. As a result, the endonucleases show inverse substrate specificity; substrates lacking a 5' end within 4 nt of the flap are cleaved poorly by Mus81-Mms4 but are cleaved well by Rad1-10. Genetically, we show that both mus81 and sgs1 mutants are sensitive to camptothecin-induced DNA damage. Further, mus81 sgs1 synthetic lethality requires homologous recombination, as does suppression of mutant phenotypes by RusA expression. These data are most easily explained by a model in which the in vivo substrate of Mus81-Mms4 and Sgs1-Top3 is a 3' flap recombination intermediate downstream of replication fork collapse.

* Corresponding author. Mailing address: Department of Molecular Biology and Biochemistry, 679 Hoes Ln., CABM, Rutgers University, Piscataway, NJ 08854. Phone: (732) 235-4197. Fax: (732) 235-4880. E-mail: brill{at}mbcl.rutgers.edu.

Molecular and Cellular Biology, May 2003, p. 3487-3496, Vol. 23, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.10.3487-3496.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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