Structure and mechanism of bacterial DNA repair pathways

暴露在DNA损伤源中会不断威胁基因组的完整性，这可能会导致遗传异常或细胞死亡。DNA双链断裂是DNA损伤最致命的形式之一，然而细胞在这种侮辱下存活下来，是保守的修复机制的结果。在某些细菌中，包括分枝杆菌、假单胞菌和芽孢杆菌，非同源末端连接修复途径通过两个关键蛋白Ku和LigD的作用来缓解DNA双链断裂。这两种蛋白质在高等真核生物中是保守的，但具有细菌所特有的独特特征。Ku和LigD负责识别、处理和连接DNA双链断裂，这是真核修复中需要多种蛋白质的功能。因此，非同源末端连接的一个关键但知之甚少的方面是，细菌中只有这两个已知蛋白质如何在分子水平上协调和执行整个修复机制。*我的研究计划的长期目标是了解细菌如何耐受和修复DNA损伤，以及这如何促进细胞存活。我的研究旨在探索对修复过程至关重要的蛋白质-蛋白质和蛋白质-核酸相互作用。为了研究这些机制，我将利用我在DNA修复和结构生物学方面的广泛背景来了解Ku-LigD相互作用如何协调从损伤识别到连接的DNA修复。我的具体目标是1)表征Ku-LigD DNA双链断裂修复复合体。通过X射线结晶学、原子力显微镜和小角X射线散射对Ku-LigD复合体的结构和生物物理表征，将为这些蛋白质在DNA损伤存在时如何相互作用提供工作模型。2)通过生化研究明确Ku刺激的LigD结扎的机制，为深入了解修复的分子机制提供依据。3)评估同源蛋白的结构和功能变异性。在细菌属之间，Ku和LigD显示出结构域的可变性；理解这种差异的功能结果将揭示细菌之间DNA修复的共同和新颖的方面。我的工作将建立Ku和LigD对细菌中非同源末端连接修复机制的基本分子理解。此外，DNA损伤经常被用于抗菌剂中，作为一种机制来清除家庭和医院表面的细菌污染，以及治疗细菌感染；然而，DNA修复会阻碍细胞死亡。因此，这项工作将确定新的目标，以改进细菌根除的方法。

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.