The long term goal of this research is to develop a detailed, 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 E. coli SOS-regulated genes dinB, which encodes a translesion DNA polymerase (DNA pol IV) that has been implicated in adaptive mutagenesis and untargeted lambda mutagenesis, and umuDC, which encodes a translesion DNA polymerase (DNA pol V) responsible for most UV and chemical mutagenesis. Following up the discovery that DinB preferentially and accurately bypasses certain N2-dG adducts, its substrate specificity will be further explored, structure-function studies carried out, and the properties of DinB compared to its mammalian and archaeal orthologs. How UmuD, UmuD', RecA and other cellular factors control the biological functions of DinB and UmuC will be investigated though a combination of biochemical, structural, and genetic approaches. The biochemical and physiological significance of NusA and beta processivity clamp interactions with DinB and UmuC with be analyzed, for example by testing whether DinB displays beta clamp-dependent dynamic processivity and whether this is modulated by RecA-mediated UmuD cleavage. The multitudinous regulatory roles of UmuD and UmuD'in controlling E. coli's responses to DNA damage will be continue to be studied, including analyzing their interactions with the catalytic and proofreading subunits of DNA pol III. Bacillus subtilis will be used as a model to investigate how cellular responses to DNA damage are coordinated within the three-dimensional architecture of intact cells, placing a special emphasis on the mismatch repair proteins, MutS and MutL, and on RecA. 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 into the origin of mutations that can contribute to cancer and aging. Our studies of E. coli DinB have revealed new properties of human DNA pol kappa and our structure-function studies of Family Y DNA polymerases should continue to offer insights into this important class of DNA polymerase. Our emphasis on the mechanisms controlling Y Family polymerases should yield new insights that are relevant to cancer and to the basis of hypermutation in the immune response.
Showing the most recent 10 out of 92 publications
