Project Summary: A major problem in medicine today is the emergence and persistence of antibiotic resistant bacteria. Although bacteria have evolved several strategies to grow in harsh environments, many bacterial species broadly cope in unfavorable conditions by regulating growth and through inducing DNA damage responses. In fact, all organisms respond to DNA damage by enlisting DNA repair pathways and by regulating cell cycle progression. Bacterial cells are constantly exposed to a broad spectrum of DNA damage caused by intracellular sources, environmental stressors, antibiotic treatments, and disinfectants applied in hospital settings. Although DNA repair and cell cycle checkpoints have been well studied in some bacteria, far less is known about these processes in Gram-positive bacteria. One major challenge is that even for the most well studied Gram-positive bacterium, Bacillus subtilis, almost half of the genes in the genome are of unknown function, representing a critical and fundamental gap in our understanding of how these bacteria mitigate stress that affects growth and proliferation. While Bacillus subtilis does not cause disease, it is closely related to a number of important human pathogens, including Methicillin-resistant Staphylococcus aureus, Listeria monocytogenes and several other pathogens that are responsible for many hospital-acquired infections, which impose significant economic burdens on our healthcare system annually. Therefore, it is important to understand how a broad group of clinically relevant bacteria respond to DNA damage and regulate cell proliferation. The long-term goal of this research is to understand the contribution of unstudied genes and novel mechanisms to DNA repair and cell cycle regulation in Gram-positive bacteria. We used large-scale genome-wide approaches to identify several uncharacterized genes that are highly conserved among Gram-positive bacteria and critical for DNA repair and regulation of cell proliferation. Two of these gene products define a new DNA excision repair pathway while four other genes are critical for DNA damage checkpoint recovery, allowing cells to re-enter the cell cycle after the damage has been repaired. We expand these experiments to continue to identify novel interactions with regulatory partners that control initiation timing and cell proliferation. We expect these studies will result in the complete mechanistic characterization of proteins involved in initiation, DNA repair, and cell cycle checkpoints. All of the genes we propose to study are either essential or cause severe growth defects when impaired, underscoring their importance as possible targets for novel antimicrobial therapies.