Characterization of mec1 Kinase-Deficient Mutants and of New Hypomorphic mec1 Alleles Impairing Subsets of the DNA Damage Response Pathway

Abstract

DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage. The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects. We show thatmec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Δ cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response. Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G₁ or during S phase. Conversely, an excess of Mec1kd in MEC1 cells does not abrogate the G₂/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable. In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G₁/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G₂. The finding that these mutants, although indistinguishable frommec1Δ cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents.

ACKNOWLEDGMENTS

We are grateful to Veronica Baldo for computer analysis. We thank J. Diffley and C. Santocanale for the antibody against Rad53, K. Nasmyth for plasmids 3746 and 3748, S. Piatti for helpful suggestions and critical reading of the manuscript, and all of the members of our laboratory for useful discussions and criticisms.

This work was supported by Telethon-Italy (grant E.1247 to M.P.L.), by a grant from the Associazione Italiana Ricerca sul Cancro and Cofinanziamento 1999 MURST-Università di Milano-Bicocca to G.L., and by CNR Target Project on Biotechnology grant CT.97.01180.PF49(F). V.P. was supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro.

V.P. and M.C. contributed equally to this work.