The responses to DNA damage are controlled by checkpoint pathways, which are conserved from yeast to human. The applicant's long-term aim is to define the function and regulation of ATR and its homologs which are involved with DMA damage checkpoints. MEC1 encodes an ATR homolog in budding yeast. ATR and Mec1 proteins are considered as damage sensor. We have shown that Mec1 interacts with Ddc2, and associates with sites of DNA damage. Our preliminary results suggest that Mec1 becomes activated after association with the site of DMA damage. Checkpoint mediators include BRCA1, 53BP1 and MDC1 in mammals and Rad9 in budding yeast. Like the mammalian mediators, Rad9 localizes to sites of DNA damage. Mammalian Chk2 kinase and its budding yeast homolog Rad53 are signal transducers. Phosphorylated Rad9 interacts with Rad53, and this Rad9-Rad53 interaction is implicated in Rad53 activation. We have shown that Mec1 phosphorylates Rad9 and promotes Rad9 association with sites of DNA damage. The DNA damage checkpoint pathway also contributes to damage repair itself. DNA polymerase C, is involved in DNA synthesis to bypass DNA damage. DNA polymerase zeta consists of Rev3 and Rev7 proteins in budding yeast. Our preliminary studies suggest that Mec1 phosphorylates Rev7 and controls its association with sites of DNA damage. From these observations, we propose to determine the mechanism of activation of the Mec1-Ddc2 complex at sites of DNA damage (Aim1), to uncover functions of phosphorylated Rad9 at sites of DNA damage (Aim2), and determine roles in Meet -dependent phosphorylation of the Rev3-Rev7 DNA polymerase (Aim 3). The failure of the checkpoint response has been implicated as a major cause of chromosomal instability, which leads to cancer in higher eukaryotes. A better understanding of DNA damage checkpoint thus should allow us to prevent the development of cancer cells.
