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

• Published in: Molecular and cellular biology 2001-06, Vol.21 (12), p.3913-3925
• Main Authors: Paciotti, Vera, Clerici, Michela, Scotti, Maddalena, Lucchini, Giovanna, Longhese, Maria Pia
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Sequence; Base Sequence; Cell Cycle - radiation effects; Cell Growth and Development; Conserved Sequence; DNA Damage; DNA Primers - genetics; DNA Repair; DNA Replication; DNA, Fungal - genetics; DNA, Fungal - metabolism; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Genes, Fungal; Intracellular Signaling Peptides and Proteins; Mec1 gene; Mec1 protein; Molecular Sequence Data; Mutagens - toxicity; Mutation; Phosphatidylinositol 3-Kinases - chemistry; Phosphatidylinositol 3-Kinases - genetics; Phosphatidylinositol 3-Kinases - metabolism; phosphatidylinositol kinase; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - radiation effects; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Ultraviolet Rays
• ISSN: 0270-7306; 1098-5549; 1098-5549
• DOI: 10.1128/MCB.21.12.3913-3925.2001

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 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 G 1 or during S phase. Conversely, an excess of Mec1kd in MEC1 cells does not abrogate the G 2 /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 G 1 /S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G 2 . 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.