DNA damage checkpoints coordinate the cellular responses to DNA damage caused by ionizing radiation and other genotoxic agents. The importance of DNA damage checkpoints for human cancer is exemplified by the cancer predisposition syndrome ataxia telangiectasia, in which a central checkpoint kinase is affected. DNA repair ensures cellular survival and genomic stability after DNA damage. Direct functional relationships between DNA damage checkpoints and DNA repair pathways have been postulated but are not well understood. The major genotoxic damage of ionizing radiation is DNA double-strand breaks. Homologous recombination is a ubiquitous, error-free DNA double-strand break repair pathway that functions also in the recovery of stalled replication forks. Aberrant processing of stalled forks is suspected to be a major source for genomic instability, a hallmark of cancer cells. How recombination is regulated is largely unknown. DNA damage-induced phosphorylation of Rad55-Rad57 is a novel checkpoint response that activates recombinational DNA repair. Rad55-Rad57 function in the assembly of the Rad51-ssDNA filament, which performs a central step during recombination but the exact mechanism is not defined. The goal is to establish mechanisms by which DNA damage checkpoints ensure survival and genomic stability after genotoxic stress. We focus on Rad55-Rad57 as a regulatory target to establish its mechanistic function and how phosphorylation enhances this function. The kinase control of Rad55-Rad57 phosphorylation suggests a potential positive feedback loop acting on the Mec1 (ATR) kinase. This hypothesis will be tested and the mechanisms involved elucidated. The evolutionary conservation of the major DNA damage checkpoint and DNA repair proteins suggests that yeast is a paradigmatic model system.
The specific aims are: (1) Establish the biological consequences of Rad55-Rad57 phosphorylation and determine the molecular mechanism of how phosphorylation affects protein stability. (2) Establish the in vivo molecular consequences of Rad55-Rad57 phosphorylation. (3) Determine the mechanism of action of Rad55-Rad57 and identify the mechanistic change imparted by phosphorylation. (4) Identify mechanisms of DNA damage checkpoint kinase regulation and test a positive feedback model involving Mec1 kinase.
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