Identifying Proteins Involved in DNA Damage Response

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DESCRIPTION (provided by applicant): DNA mismatch repair (MMR) plays an important role in maintaining genomic stability as defects in MMR lead to the development of cancer. In the past, the genome maintenance function of MMR had been attributed to its ability to correct mismatches. However, increasing evidence suggests that the MMR system also maintains genomic stability by promoting apoptosis in response to DNA damage induced by physical and chemical agents, including environmental chemical carcinogens. This response is known to be dependent on MutS and MutL homologs, and eliminates potentially carcinogenic cells from growing. The MMR-dependent apoptosis appears to involve a signaling network, which transmits a DNA damage signal to the apoptotic machinery to kill damaged cells. However, how the network works and what proteins are involved in this network are poorly understood. This study aims to identify and characterize proteins that participate in MMR-dependent apoptosis induced by environmental chemical carcinogens using a prote0mic approach. First, MMR-proficient and deficient cells will be treated with chemical carcinogens, and protein expression profiles before and after treatments from these cells will be analyzed by 2-dimensional gel electrophoresis. Proteins with differential expression will be subjected to mass spectromic analysis, and the resulting peptides will be used for database homology searching to reveal their identities. Second, given the involvement of protein phosphorylation or dephosphorylation in many signaling networks, we hypothesize that many proteins participating in MMR-dependent apoptosis may be phosphorylated or dephosphorylated. To test this hypothesis, proteins that are identified in this study and known previously to be involved in MMR-dependent apoptosis will be analyzed for their phosphorylation status by 1-dimensional or 2-dimensional gels combined with Western blot analysis, followed by mass spectrometry analysis. Data resulting from this project will formulate the basis for more detailed investigations (R01 applications) to fully understand the molecular mechanism by which the MMR system maintains genomic stability by promoting apoptosis induced by environmental chemical carcinogens. Because certain cancer chemotherapeutics, e.g., cisplatin and alkylating agents, can signal apoptosis in an MMR-dependent fashion, this study will also impact cancer treatment, particularly cancers caused by MMR defects.