Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway

Abstract

Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 playsimilar roles in the DNA damage checkpoint control in budding yeast.These genes are required for DNA damage-induced Rad53 phosphorylationand considered to function upstream of RAD53 in the DNA damage checkpointpathway. Here we identify Mec3 as a protein that associates withRad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3interact physically in vivo. The amino terminus of Rad17 is requiredfor its interaction with Mec3, and the protein encoded by the rad17-1allele, containing a missense mutation at the amino terminus, isdefective for its interaction with Mec3 in vivo. Ddc1 interacts physicallyand cosediments with both Rad17 and Mec3, indicating that these threeproteins form a complex. On the other hand, Rad24 is not found toassociate with Rad17, Mec3, and Ddc1. DDC1 overexpression can partiallysuppress the phenotypes of the rad24Delta mutation: sensitivity toDNA damage, defect in the DNA damage checkpoint and decrease in DNAdamage-induced phosphorylation of Rad53. Taken together, our resultssuggest that Rad17, Mec3, and Ddc1 form a complex which functionsdownstream of Rad24 in the DNA damage checkpoint pathway.