The way organisms respond to radiation exposure is important since induced DNA lesions misrepaired or left unrepaired can lead to death, mutation or cancer. Previous efforts have focused on fission yeast S. pombe rad9, a gene that promotes gamma-ray resistance, UV-resistance, resistance to the DNA replication inhibitor hydroxyurea (flU), and early S phase as well as G2 checkpoint control. Recently, human (HRAD9) and mouse (Mrad9) genomic and cDNA orthologues have been identified and the cDNAs were found to partially complement several defects demonstrated by rad9::ura4+ yeast. Furthermore, we found that HRAD9 protein binds the checkpoint proteins HHUS1 and HRAD1 at its C-terminal region, and contains a BH3-like domain at its N-terminal region that can bind the anti-apoptotic proteins BCL-2 and BCL-xL, and can cause apoptosis when overexpressed. We also found that this multifunctional protein can bind p53 and co-regulate p21. The major focus of this proposal builds on and extends a large amount of data accrued with yeast and mammalian systems, and centers on the function of Mrad9 in mammals. Specifically, this proposal will make use of Mrad9knockout cells and mice to address the hypothesis that, like the yeast gene, Mrad9plays a key role in the response of cells to DNA damage or inhibition of DNA replication. A working model to at least partly explain RAD9 function in cell cycle progression and/or apoptosis, and aspects of which will be addressed, is the following: after DNA damage, ATM mediates phosphorylation of RAD9 (and p53 on serine-15), that regulates p21 levels; increased p21 abundance inhibits Rb phosphorylation, which is in a form unable to activate E2F1, and thus transcription of multiple genes involved in cell cycle progression, apoptosis and related processes are not induced. Experimental goals are 1) construct Mrad9knockout cells and mice; 2) characterize the biological impact of Mrad9mutations at the cellular level, including sensitivity to DNA damaging agents, checkpoint control and apoptosis; 3) characterize the molecular impact of Mrad9 mutations by examining changes in the status of proteins in the working model; 4) address the epistatic relationship between Mrad9 and ATM, p2] and p53 in terms of biological and molecular impact; 5) perform structure/function studies to define Mrad9regions important for activity; 6) assess the role of Mrad9in tumorigenesis; and 7) follow up preliminary results indicating that Mrad9 participates in embryonic development, or organ maintenance/development and its relation to the role of the protein in response to DNA damage. These studies will examine the function of Mrad9 from molecular to cellular levels, and in whole animals, and could impact on radiotherapy and on understanding its potential as a genetic marker indicative of susceptibility to DNA damage.
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