Evolution of Function of Bacterial Nucleoid-Associated Proteins

该奖项支持专注于了解重要蛋白质在细菌细胞结构和功能中的作用的研究。DNA的大小要求它被压缩到适合细胞的尺寸，同时仍然允许它与细胞蛋白质接触。细菌基因组与许多不同的DNA结合蛋白有关，包括被称为HU的蛋白。除了参与DNA的组织外，Hu蛋白还参与调节依赖DNA的过程，如复制、修复和基因表达，因此对正常的细胞功能至关重要。在这个项目中，重点将放在耻垢分枝杆菌编码的Hu变异体上。分枝杆菌编码一种不同寻常的双域HU蛋白。双域HU蛋白(通常称为HLP，类似组蛋白的蛋白质)的有趣之处在于，它们似乎只由特别擅长抵抗环境压力的细菌物种编码。例如，据报道，对于分枝杆菌，HLP在休眠期间更加丰富。这项研究的目的是确定耻垢分枝杆菌HLP的功能作用，特别关注富含赖氨酸的延伸的作用。了解分枝杆菌HLP的功能作用将阐明分枝杆菌生理学的一个独特方面，以及编码两个结构域HU同源物的其他细菌物种的特定需求。一项旨在确定HLP在DNA紧凑中的作用的DNA结合分析，以及旨在确定HLP保护DNA免受损害的能力的分析正在计划中。耻垢分枝杆菌还编码一种名为Ku的蛋白质，该蛋白质有望在DNA双链断裂的修复中发挥作用；Ku与HLP具有富含赖氨酸延伸的关键特征。这一共有的序列特征将被用来确定富含赖氨酸的结构域在这两种蛋白质的DNA末端连接中的作用，并确定它们在双链断裂修复中的作用。最终的目标是确定HLP更加丰富的条件，并研究HLP缺失对全球基因表达的影响。这条调查路线有望确定需要过量HLP的条件，以及HLP在DNA组织、DNA保护和DNA双链断裂修复中的作用。这个项目将通过为研究生和本科生提供机会，获得与蛋白质-蛋白质和蛋白质-核酸相互作用分析相关的技术方面的研究经验和熟练程度，从而将研究和教育结合起来。将强调少数民族学生的参与，预计各级学生将在全国会议上传播他们的研究成果。

This award supports research focused on understanding the role of important proteins in the structure and function of a bacterial cell. The size of the DNA requires that it be compacted to fit the dimensions of the cell, while still allowing its accessibility to cellular proteins. The bacterial genome is associated with a number of different DNA-binding proteins, including proteins known as HU. Aside from being involved in the organization in DNA, HU proteins function in regulating DNA-dependent processes, such as replication, repair and gene expression and are therefore critical for normal cellular function. In this project, the focus will be on the HU variant encoded by Mycobacterium smegmatis. Mycobacteria encode an unusual two-domain HU protein. What is intriguing about two-domain HU proteins (typically referred to as Hlp, for Histone Like Protein) is that they appear to be encoded only by bacterial species that are particularly adept at resisting environmental stress. For mycobacteria, Hlp has, for example, been reported to be more abundant during dormancy. The goal of this research is to define the functional role of M. smegmatis Hlp, with a specific focus on the role of the lysine-rich extension. Understanding functional roles of mycobacterial Hlp will illuminate a unique aspect of mycobacterial physiology as well as the specific demands of other bacterial species that encode two-domain HU homologs. A combination of DNA-binding assays designed to determine the role of Hlp in DNA compaction are planned, as well as assays aimed at determining the ability of Hlp to protect DNA from damage. M. smegmatis also encodes a protein named Ku, which is expected to function in repair of DNA double strand breaks; Ku shares with Hlp key features of the lysine-rich extension. This shared sequence feature will be exploited to determine the role of the lysine-rich domain in DNA end-joining by both proteins and to ascertain their roles in double strand break repair. A final aim is centered on determining conditions under which Hlp is more abundant and on investigating consequences of Hlp deletion on global gene expression. This line of inquiry is expected to define conditions under which excess Hlp is required and the roles of Hlp in DNA organization, DNA protection, and DNA double strand break repair. This project will integrate research and education by offering the opportunity for both graduate and undergraduate students to acquire research experience and proficiency in techniques associated with analysis of protein-protein and protein-nucleic acid interactions. The involvement of minority students will be emphasized, and students at all levels will be expected to disseminate their research at national meetings.