Srs2 and Sgs1 DNA Helicases Associate with Mre11 in Different Subcomplexes following Checkpoint Activation and CDK1-Mediated Srs2 Phosphorylation

Abstract

Mutations in the genes encoding the BLM and WRN RecQ DNA helicases and the MRE11-RAD50-NBS1 complex lead to genome instability and cancer predisposition syndromes. The Saccharomyces cerevisiae Sgs1 RecQ helicase and the Mre11 protein, together with the Srs2 DNA helicase, prevent chromosome rearrangements and are implicated in the DNA damage checkpoint response and in DNA recombination. By searching for Srs2 physical interactors, we have identified Sgs1 and Mre11. We show that Srs2, Sgs1, and Mre11 form a large complex, likely together with yet unidentified proteins. This complex reorganizes into Srs2-Mre11 and Sgs1-Mre11 subcomplexes following DNA damage-induced activation of the Mec1 and Tel1 checkpoint kinases. The defects in subcomplex formation observed in mec1 and tel1 cells can be recapitulated in srs2-7AV mutants that are hypersensitive to intra-S DNA damage and are altered in the DNA damage-induced and Cdk1-dependent phosphorylation of Srs2. Altogether our observations indicate that Mec1- and Tel1-dependent checkpoint pathways modulate the functional interactions between Srs2, Sgs1, and Mre11 and that the Srs2 DNA helicase represents an important target of the Cdk1-mediated cellular response induced by DNA damage.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank Michele Giannattasio and Marco Muzi-Falconi for providing the yeast strain containing the DNA library for the two-hybrid screening and for technical suggestions. We also thank the Immunological Services at IFOM, Anastasia Baryshnikova and the all members of our lab, for stimulating discussion and reagents, and Achille Pellicioli and Hannah Klein for critical reading of the manuscript.

This work was supported by the Associazione Italiana per la Ricerca sul Cancro, the European Union (grant RTN2-2001-00177), and partially by Telethon (grant GGP030412) and the Ministero della Salute. I.C. is supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro.