This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research;2) exposing undergraduates to research;and 3) strengthening the research environment in non-research intensive universities. The long term goal of this research is to understand the mechanisms by which cells respond to DNA damage. The link between DNA damage and human health is well-established: the inability to correctly repair damaged DNA can result in various forms of colon, breast, and ovarian cancers in humans. This project focuses on the translesion synthesis SOS response to DNA damage, which is accomplished by Y-family polymerases (Cordonnier &Fuchs 1999). The existence of similar, extensive DNA damage sensing and repair mechanisms in organisms ranging from humans to bacteria demonstrates this central role of DNA integrity. Homologs to bacterial Y-family polymerases exist in humans (e.g. pol 7) and other eukaryotes (polymerase Rev1p and Rad30p) (McDonald et al. 1999;Goodman 2002;Nohmi 2006), where mutations in pol 7 predispose humans to skin cancer (Cleaver 2000). In a typical bacterial SOS response pathway, after DNA damage, the UmuD protein is induced, self-cleaves, and then acts with its partner, UmuC, to function as a translesion synthesis DNA polymerase. To accomplish our overall goals, we will establish how an unusual homolog of UmuD present in the ubiquitous soil bacterium Acinetobacter baylyi strain ADP1 functions after DNA damage. Both the genetics and functioning of the response to DNA damage in ADP1 is unlike other bacteria: the umuC gene is interrupted and incomplete. Furthermore, the UmuD of ADP1 (UmuDAb) is unique: it contains an extra N-terminal region, and it regulates the expression of a DNA damage-inducible gene, ddrR. We propose two hypotheses for UmuDAb involvement in the DNA damage response of ADP1: (A) UmuDAb can behave like other UmuD proteins, cleaving itself after DNA damage and acting with UmuC, and (B) The """"""""extra"""""""" N-terminal region of UmuDAb is required for its action as a regulator. To test these hypotheses, our aims will be to determine: (i) if UmuDAb is activated for self- cleavage by DNA damage, using Western blotting analyses of UmuDAb (or UmuDAb fragments) after exposure of ADP1 cells to DNA damaging chemicals, (ii) if UmuDAb can interact with an exogenous UmuC to conduct translesion DNA synthesis or checkpoint functions, by complementing either an Escherichia coli umuD- mutant with the ADP1 umuDAb allele, or the ADP1 strain with the E. coli umuC, and (iii) if the """"""""extra"""""""" N-terminal region of UmuDAb is required for its regulation of ddrR, by measuring the inducible expression of a ddrR::lacZ gene fusion in an ADP1 strain with a mutant umuDAb allele lacking the extra N- terminal domain, This research is significant because it will enhance our understanding of the ways in which cells can achieve DNA repair, specifically, with the SOS response. This project will provide research training opportunities for Morehead State University undergraduates, 66% of whom grew up in Appalachia and have largely not been exposed to, nor ever engaged in, scientific research. The increased research infrastructure, expectation of and opportunities for student engagement in authentic research will benefit the entire biology department and Morehead State University.
To enhance our understanding of the mechanisms that human as well as bacterial cells use to repair their DNA, this research will characterize mechanisms of action of an atypical bacterial DNA damage response protein that is a component of an error-prone polymerase. Similar polymerases exist in bacteria, yeast and humans, with defects in these polymerases causing human cancer after exposure to ultraviolet light. This project has additional biomedical implications for bacterial disease, as the SOS response in bacteria can be induced by antibiotic treatment and lead to the spread of antibiotic resistance and other virulence genes among bacteria.
