Role of the C terminus of Mec1 checkpoint kinase in its localization to sites of DNA damage.
The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), coordinate the cellular response to DNA damage. In budding yeast, ATR homologue Mec1 plays a central role in DNA damage signaling. Mec1 interacts physically with Ddc2 and functions in the form of the Mec1-Ddc2 complex. To identify proteins interacting with the Mec1-Ddc2 complex, we performed a modified two-hybrid screen and isolated RFA1 and RFA2, genes that encode subunits of replication protein A (RPA). Using the two-hybrid system, we found that the extreme C-terminal region of Mec1 is critical for RPA binding. The C-terminal substitution mutation does not affect the Mec1-Ddc2 complex formation, but it does impair the interaction of Mec1 and Ddc2 with RPA as well as their association with DNA lesions. The C-terminal mutation also decreases Mec1 kinase activity. However, the Mec1 kinase-defect by itself does not perturb Mec1 association with sites of DNA damage. We also found that Mec1 and Ddc2 associate with sites of DNA damage in an interdependent manner. Our findings support the model in which Mec1 and Ddc2 localize to sites of DNA damage by interacting with RPA in the form of the Mec1-Ddc2 complex.
Nakada
Daisuke
D
Department of Cell Biology and Molecular Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.
Hirano
Yukinori
Y
Tanaka
Yuya
Y
Sugimoto
Katsunori
K
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C440014:LCD1 protein, S cerevisiae; D010750:Phosphoproteins; C082728:RFA1 protein, S cerevisiae; C082727:RFA2 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases