This AREA R15 grant will (i) support meritorious research, (ii) strengthen the research environment of a non-research intensive university, and (iii) engage undergraduate students in biomedical research. The overall goal of this research is to understand the mechanisms used by cells to respond to DNA damage, which is important because defects in DNA repair processes are linked to various cancers in humans, but their use can cause antibiotic resistance in bacteria. Control of DNA damage repair processes thus must allow DNA repair processes to be induced appropriately, while confining their use to needed situations. Specifically, this research will identify how the mechanism of a error-prone polymerase manager (UmuD) homolog, which has evolved to repress DNA damage response (SOS) genes, is affected by the DdrR protein in the Acinetobacter bacterial genus. Mechanisms of this novel regulator, UmuDAb, will be dissected in the opportunistic pathogen Acinetobacter baumannii. DNA damage causes antibiotic resistance in this bacterium via the induction and SOS mutagenesis action of its numerous umuDC genes, which are normally repressed by UmuDAb. The UmuDAb regulon includes all six error-prone polymerase umuDC genes, umuDAb itself, and the ddrR gene transcribed divergently from umuDAb. Our preliminary data indicate that, like UmuDAb, DdrR also regulates the DNA damage- induced umuDC genes as well as umuDAb and ddrR. We hypothesize that UmuDAb regulatory actions (repression and self-cleavage) require cooperation with DdrR action.
The specific Aims of this project are to: 1) Detect UmuDAb and DdrR co-regulation of DNA damage-induced genes, using transcriptome analyses of a ddrR mutant; 2) Identify how UmuDAb regulatory actions of DNA binding and self-cleavage are impacted by DdrR, measuring the expression and SOS mutagenesis phenotypes of error-prone polymerase target genes in ddrR and umuDAb mutant strains; and 3) Determine if UmuDAb and DdrR physically interact to achieve co-regulation of DNA damage responses, using ChIP-Seq, two-hybrid analyses, cross-linking, and biochemical approaches. This project will create new knowledge about the regulatory mechanisms and proteins that bacteria use to respond to DNA damage by investigating both the novel UmuDAb repressor and the DdrR co-repressor. We will identify how the bacteria integrate UmuDAb and DdrR co-regulation of the mutagenic polymerases, which could reveal methods for preventing drug resistance in pathogens. Experiments will be completed with undergraduate and graduate students 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, and the expectation of and opportunities for student engagement in research, will be beneficial to the entire Biology and Chemistry department at Morehead State University.
Both humans and bacteria respond to DNA damage using similar error-prone (mutagenic) DNA polymerases, where polymerase defects can cause cancer after exposure to DNA damage, and polymerase action can cause mutations in bacterial pathogens that lead to antibiotic resistance and their increased virulence. This project will identify the mechanism of action of a novel bacterial regulator, which closely resembles a human error-prone DNA polymerase, and its co-repressor. These experiments will help identify how pathogens control mutagenic responses to DNA damage that is present in a clinical setting (e.g., the UV light used to disinfect hospital surfaces, or antibiotic treatment).
