PROJECT SUMMARY/ABSTRACT Our environment is replete with agents, such as high energy radiation, air pollutants, industrial chemicals, pesticides, and heavy metals that can damage DNA and thus compromise genome stability. Among myriad environmentally-induced DNA lesions, the DNA double-strand break (DSB) is possibly the most deleterious because of its potential to induce chromosomal rearrangements and collapse of DNA replication forks. Homologous Recombination (HR) is a conserved mechanism that eliminates DSBs in an error-free fashion, and defects in HR are very mutagenic, and associated with premature aging, neurological disorders, and cancer. As in other complex biological processes, HR is subject to regulatory controls being dependent on protein phosphorylation-dephosphorylation. Of all known phosphorylation-dephosphorylation circuitries within the DNA damage response (DDR), the kinases and phosphatases that act on tyrosine residues in target proteins remain the most poorly defined. Importantly, we have found that the dual activity phosphatase EYA4 can dephosphorylate physiologically relevant tyrosine residues on the DDR factors H2AX and RAD51. Accordingly, depleting EYA4 sensitizes cells to ionizing radiation and genotoxic chemicals, and it also leads to severe destabilization of the genome, causing chromosomal breakage and high levels of aneuploidy. Unexpectedly, our biochemical analysis of highly purified EYA4 has shown that its protein phosphatase activity is dependent on DNA addition and that EYA4 has intrinsic DNA binding activity. Moving forward, we will use a combination of biochemistry, cell biology, and NMR to understand how the DNA binding activity of EYA4 contributes to its role in DNA damage repair and genome protection, at DSBs and also DNA replication forks. Through these studies, we will gain insight into a hitherto unknown circuitry of DDR protein phosphorylation-dephosphorylation that has a major impact on the cellular response to environmental exposures.