Structure and mechanism of bacterial DNA repair pathways

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Exposure to sources of DNA damage continually threatens genomic integrity, which can result in genetic aberrations or cell death. DNA double-strand breaks are one of the most lethal forms of DNA damage, however cells survive such insults as the result of conserved repair mechanisms. In certain bacteria, including Mycobacterium, Pseudomonas and Bacillus, the non-homologous end joining repair pathway mitigates DNA double-strand breaks through the actions of two critical proteins Ku and LigD. These two proteins are conserved in higher eukaryotes but have distinguishing features unique to bacteria. Ku and LigD are responsible for recognizing, processing and ligating the DNA double-strand break, functions that require multiple proteins in eukaryotic repair. Therefore, a critical yet poorly understood aspect of non-homologous end joining is how only these two known proteins in bacteria coordinate and execute the entire repair mechanism at the molecular level. The long-term goal of my research program is to understand how DNA damage is tolerated and repaired in bacteria, and how this promotes cell survival. My research aims to probe the protein-protein and protein-nucleic acid interactions that are critical to the repair process. To investigate these mechanisms, I will leverage my extensive background in DNA repair and structural biology to understand how the Ku-LigD interactions coordinate DNA repair from damage recognition to ligation. My specific objectives are to 1) Characterize the Ku-LigD DNA double-strand break repair complex. Structural and biophysical characterization of the Ku-LigD complex by x-ray crystallography, atomic force microscopy and small-angle x-ray scattering will provide working models of how these proteins interact in the presence of DNA damage. 2) Define the mechanism of Ku-stimulated ligation by LigD through biochemical studies that will provide insight into the molecular mechanism of repair. 3) Evaluate structural and functional variability of homologous proteins. Between bacterial genera, Ku and LigD display domain variability; understanding the functional outcomes of this divergence will reveal shared and novel aspects of DNA repair among bacteria. My work will establish a fundamental molecular understanding of the non-homologous end joining repair mechanism in bacteria by Ku and LigD. Furthermore, DNA damage is often used in antimicrobials as a mechanism to remove bacterial contamination from surfaces in homes and hospitals, as well as to treat bacterial infections; yet DNA repair hinders cell death. Therefore, this work will identify new targets to improve methods of bacterial eradication.