Mechanisms of DNA Repair and DNA Damage Dependent Cell Cycle Control in Bacillus subtilis

[摘要] The DNA damage response is a conserved process found in all domains of life that serves to protect the genetic material of living organisms. When an organism encounters DNA modifications or ;;DNA damage,’ a response is elicited resulting in increased levels of DNA repair proteins and activation of a cell cycle checkpoint. In bacteria the DNA damage response has been studied extensively in Escherichia coli. Although the activation of the DNA damage response is conserved in many bacteria, the downstream steps of DNA repair and cell cycle regulation diverge significantly. Therefore, exactly how bacteria respond to DNA damage is still not clear. I used forward genetics in the model organism Bacillus subtilis to screen for non-essential genes that are required for mitigating the toxicity of three distinct classes of DNA damage inducing drugs. I identified two proteases that function in the DNA damage checkpoint. Through studies employing genetics, cell biology, and biochemical approaches, I present that these two proteases degrade the cell division inhibitor that is expressed as part of the DNA damage response. Thus, these two proteases aid in establishing the level of cell division inhibitor required for checkpoint activation, while also serving to clear excess inhibitor preventing further checkpoint activation. Additionally, a detailed investigation using genetics and cell biology uncovered a DNA damage checkpoint antagonist that functions by preventing the cell division inhibitor from activating the checkpoint. Therefore, the checkpoint antagonist aids in establishing the threshold for checkpoint activation along with the checkpoint proteases. The genetic screens also revealed a putative DNA repair pathway. Two genes in a putative operon encoding a helicase and an exonuclease were important for DNA damage caused by a single DNA damaging agent. Genetic analyses indicate that these genes operate as part of a novel nucleotide excision repair pathway that appears to be specific to a natural antibiotic. Finally, I studied a protein that is annotated as a potential mismatch repair protein, yet had no detectable mismatch repair phenotype. My investigation demonstrated that this protein functions by promoting homologous recombination, likely by acting as a secondary Holliday junction endonuclease. Together, my studies have uncovered new pathways of DNA repair and cell cycle regulation in the DNA damage response in bacteria. Further, in each of my investigations I identified at least two ways of performing a single function in the DNA damage response, uncovering a more general strategy of favoring a robust DNA damage response in B. subtilis.

[发布机构] University of Michigan

[效力级别] DNA repair

[关键词] Bacillus subtilis;DNA repair;checkpoint;protease;CRISPR/Cas9;Molecular;Cellular and Developmental Biology;Science;Molecular, Cellular, and Developmental Biology