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Molecular and Cellular Biology, June 2001, p. 3913-3925, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3913-3925.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Vera Paciotti, Michela Clerici, Maddalena Scotti, Giovanna Lucchini, and Maria Pia Longhese*

Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, 20126 Milan, Italy

Received 13 November 2000/Returned for modification 16 March 2001/Accepted 28 March 2001

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 that mec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Delta 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 from mec1Delta 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.

* Corresponding author. Mailing address: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy. Phone: 39-02-64483425. Fax: 39-02-64483565. E-mail: mariapia.longhese{at}unimib.it.

Molecular and Cellular Biology, June 2001, p. 3913-3925, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3913-3925.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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