Molecular Mechanism of Error-free DNA Damage Response

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DNA-based organisms face a great challenge of maintaining genomic stability, due to DNA damage that arises constantly by cellular metabolic processes or by environmental conditions (such as UV and ionizing radiation and chemical agents). A variety of DNA damage responses have evolved to deal with DNA damage and genomic alterations; these responses include DNA repair and lesion bypass. Most lesions (damage) are repaired by DNA repair. However, some lesions are resistant to DNA repair, become blocking sites for normal DNA replication, and pose serious problems for cell survival. Lesion bypass pathways are required for cell survival. The DNA damage tolerance process is divided into two parallel pathways: error-prone and error-free lesion bypasses. The error-free pathway bypasses DNA lesions, without increasing the mutation rates. A little knowledge exists for the error-free pathway. Homologous recombination (HR) is required for error-free lesion bypass, but the molecular events involved are not clear. Recently, four genes in budding yeast, CSM2, PSY3, SHU1 and SHU2, have been identified that are involved in the error-free lesion bypass. The four gene products form a stable, 4-subunit Shu complex that is required for efficient HR. Inactivation of any of these genes makes yeast cells more sensitive to DNA damage agents. The Shu complex binds both single- and double-stranded DNA and appears to recruit HR proteins to facilitate DNA strand switching. Thus, the Shu complex is a regulator of DNA recombination or exchange and important to error-free DNA lesion bypass. In addition, Shu homologs have been identified in fission yeast, C. elegans and humans, indicating a common mechanism has been conserved in eukaryotes. To reveal the molecular mechanism of error-free lesion bypass, we propose to conduct a structure-function study of the Shu complex. The current goals of this study are: 1) determine the biochemical functions of the Shu complex; 2) determine the structures of the Shu complex in binary (with ATP, or DNA) and ternary forms (with both ATP and DNA) forms; 3). determine the structure of the human Shu homolog SWS1-SWAP1. We will use X-ray crystallography analysis, in combination with molecular biology and biochemistry techniques, to study the tetrameric complex. The proposed studies of the Shu complex will help us to determine which Shu proteins manipulate the specificity of DNA binding and how the Shu proteins cooperate to facilitate HR and error-free DNA lesion bypass.