The long term goal of this research is to develop a detailed and integrated view of how organisms respond to damage to their genetic material. The proposed multidisciplinary research program places a particular emphasis on the roles of the SOS-regulated umuDC genes, which play roles in a DNA damage checkpoint and also encode the DNA polymerase (DNA po1 V) that acts in translesion synthesis, the molecular basis of most UV and chemical mutagenesis. It also includes analyses of the interactions and functions of the SOS-regulated DinB (DNA pol IV) protein and a new initiative to use the techniques of prokaryotic cell biology to determine how the subcellular organization of DNA repair and mutagenesis proteins changes in response to DNA damage. Our work on the structure of the various forms of the umuD gene product has already offered important insights into its mechanism of action and we will continue to investigate the structures of the umuDC and dinB gene products and explore structure-function relationships. To test our hypothesis that UmuD and UmuD' form part of a higher order regulatory system that controls the events that occur when DNA po1 III encounters a lesion, we will characterize the interactions of the umuDC gene products with subunits of DNA po1 III and other cellular proteins. To further our understanding of the molecular mechanisms involved in umuDC-dependent checkpoint control and umuDC-dependent translesion synthesis, we will carry out physiological experiments that test various hypotheses concerning umuDC function. We will identify proteins that interact with DinB (DNA po1 IV) and investigate its physiological role(s). We will carry out cell biological experiments designed to characterize umuC homologs in B. subtilis and determine their subcellular organization, to examine the effects of DNA damage on DNA replication factories, and examine the subcellular organization of other DNA repair systems. Since the process of translesion synthesis appears to be a universal process by which mutations are introduced as a consequence of DNA damage, further studies of the roles of the umuDC gene products in this process should yield insights that are relevant to the origin of cancer and aging. They should also be relevant to certain other human diseases and to evolution. Insights into the structure of UmuC-related DNA polymerases should help us understand the role of the Xeroderma pigmentosum variant (XP-V) product in cancer prevention. Our proposed research may also lead to the identification of new targets for drug development.
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