Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. This AREA R15 renewal will accomplish the objectives of (i) supporting meritorious research, (ii) strengthening the research environment of non-research intensive universities, and (iii) engaging undergraduate students in biomedical research. The overall goal of this research is to understand how cells respond to DNA damage, because use of these pathways cause mutations in bacteria that drive increased virulence and drug resistance, whereas defects in this pathway can result in various cancers in humans. This proposal will characterize the unique mechanisms of a translesion synthesis protein (UmuD) homolog that regulates DNA damage response genes. The bacterial UmuD?2C Y-family polymerase is induced after DNA damage via relief of LexA repression and conducts mutagenic, translesion DNA synthesis. Homologs of bacterial Y-family polymerases exist in eukaryotes, where mutations in pol h predispose humans to skin cancer. Unlike other bacteria, non-enteric Acinetobacter baylyi possesses an operon consisting of a umuD homolog, umuDAb, and a truncated, incomplete umuC. The genotypic and phenotypic characteristics of UmuDAb constitute a chimera of LexA and UmuD: it has an extended N-terminal domain (NTD) like LexA, and a C-terminal domain (CTD) conducting self-cleavage similar to both UmuD and LexA. Furthermore, umuDAb cannot complement an Escherichia coli umuD mutant, but rather regulates ddrR and itself in response to DNA damage. We hypothesize that UmuDAb is a LexA-like repressor. The mechanisms of UmuDAb action will be dissected in the translesion-synthesis incompetent A. baylyi as well as in the opportunistic pathogen Acinetobacter baumannii, which conducts UV-induced mutagenesis and has both the ddrR-umuDAb locus and an E. coli-like umuDC operon.
The specific Aims of this project are to: (1) Delineate the actions of the C- and N-terminal domains of UmuDAb that are required for regulation of genes after DNA damage;(2) Describe the functional roles and regulatory relationship of the multiple umuD alleles of A. baumannii in its response to DNA damage.
These Aims will be accomplished through experiments assaying ddrR regulation and UV-induced mutagenesis ability in cells with mutations in the N- or C-terminal domains of UmuDAb. Transcriptome analyses will complement these experiments. This project will extend our knowledge of the regulation and function of Y-family polymerases as well as their role in causing mutations in bacterial pathogens. These experiments will be completed by undergraduates mentored and trained by the PI to perform authentic biomedical research, help write scientific manuscripts, and communicate their findings to the campus community and larger scientific networks. The enhanced research infrastructure, as well as the expectation of and opportunities for student engagement in research, will be beneficial to the entire Biology and Chemistry department at Morehead State University.
This project investigates common mechanisms that human as well as bacterial cells use to repair their DNA by characterizing the action of a novel bacterial regulatory protein that is a homolog of a human error-prone DNA polymerase component. Similar polymerases exist in bacteria, yeast and humans, where defects in these polymerases can cause cancer after exposure to DNA damage. We will extend our previous work in Acinetobacter baylyi to the multi-drug resistant pathogen Acinetobacter baumannii, which will help determine how the DNA damage response to antibiotic treatment of bacteria leads to the spread of antibiotic resistance and other virulence genes among bacteria.
|Witkowski, Travis A; Grice, Alison N; Stinnett, DeAnna B et al. (2016) UmuDAb: An Error-Prone Polymerase Accessory Homolog Whose N-Terminal Domain Is Required for Repression of DNA Damage Inducible Gene Expression in Acinetobacter baylyi. PLoS One 11:e0152013|
|Hare, Janelle M; Ferrell, Joshua C; Witkowski, Travis A et al. (2014) Prophage induction and differential RecA and UmuDAb transcriptome regulation in the DNA damage responses of Acinetobacter baumannii and Acinetobacter baylyi. PLoS One 9:e93861|
|Hare, Janelle M; Bradley, James A; Lin, Ching-li et al. (2012) Diverse responses to UV light exposure in Acinetobacter include the capacity for DNA damage-induced mutagenesis in the opportunistic pathogens Acinetobacter baumannii and Acinetobacter ursingii. Microbiology 158:601-11|
|Hare, Janelle M; Adhikari, Sabal; Lambert, Kasandra V et al. (2012) The Acinetobacter regulatory UmuDAb protein cleaves in response to DNA damage with chimeric LexA/UmuD characteristics. FEMS Microbiol Lett 334:57-65|