Grant: Discovery Grants Program - Individual

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(removed) by DNA repair systems. 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. Lesion bypass is a DNA-damage tolerance process that replicates through the replication-blocking DNA lesions without removal of the lesions and prevents damage-induced cell death. The DNA damage tolerance process is subdivided into two parallel pathways: error-prone and error-free lesion bypasses. The error-prone one uses special proteins called low-fidelity translesion DNA polymerases to carry out direct replication over the damaged DNA template and bypass lesions, but at a cost of increased mutation rates. The error-free pathway bypasses DNA lesions without increasing the mutation rates. While error-prone lesion bypass has been well characterized, less knowledge exists for the error-free pathway. Homologous recombination (HR) or DNA exchange 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 DNA strand exchange) and important to error-free DNA lesion bypass. 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) to determine the interactions between the Shu complex and different DNA structures, 2) to determine the structures of the Shu complex in different assembly forms, 3) to determine the structures of the Shu complex in DNA-bound forms. We will use X-ray crystallography analysis, in combination with molecular biology and biochemistry techniques, to study the Shu complex. The proposed studies will help us to determine which Shu proteins manipulate the specificity of DNA binding and how the four Shu proteins cooperate to facilitate HR-coupled error-free DNA lesion bypass.