Evolution of Function of Histone-like HU Proteins

细菌组蛋白样蛋白HU已在大肠杆菌中得到初步鉴定。除了在基因组DNA紧凑中发挥作用外，E.coliHu还被证明参与了DNA修复和重组等过程。然而，体外归因于HU直系物的DNA结合特性的范围表明，它们在体内发挥着不同的作用。实现了一种识别底物专一性的分子决定因素的比较方法。假设是，HU蛋白强加的两个扭结是必须确保的‘铰链’，只有当稳定的接触不足以确保完美双链DNA中的铰链时，才会倾向于扭曲的DNA。定点突变和电泳技术将被用来测量结合亲和力、底物专一性和位点大小。蛋白质将包括来自枯草芽孢杆菌和高温嗜热菌Thermotoga maritima的HU。这一假设还将被认为，DNA包裹在HU蛋白表面需要破坏表面盐桥。这一假设将使用由芽孢杆菌噬菌体编码的序列特异性同源物HU同源TF1和来自Maritima的HU进行实验评估，方法是系统地修改带电表面残基并测量结合常数及其对盐的依赖。该专业致力于将研究和教学结合起来，为研究生和本科生提供机会，让他们获得与蛋白质-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。研究生将是开展这些研究项目不可或缺的一部分，少数族裔本科生将继续为相关项目做出贡献。

The bacterial histone-like protein HU has been primarily characterized in Escherichia coli. In addition to its role in genomic DNA compaction, E. coli HU has also been shown to participate in processes such as DNA repair and recombination. However, the range of DNA-binding properties ascribed to HU orthologs in vitro suggest that they serve distinct roles in vivo. A comparative approach to identifying molecular determinants of substrate specificity is implemented. The hypothesis is that the two kinks imposed by HU proteins are 'hinges' that must be secured, and that preference for distorted DNA occurs only if stabilizing contacts are insufficient to secure the hinges in perfect duplex DNA. Site-directed mutagenesis and electrophoretic techniques will be used to measure binding affinity, substrate specificity and site size. Proteins will include HU from Bacillus subtilis and from the hyperthermophile Thermotoga maritima. The hypothesis will also be considered that wrapping of DNA about the surface of HU proteins requires disruption of surface salt bridges. This hypothesis will be evaluated experimentally using the HU homolog TF1, a sequence-specific homolog encoded by a Bacillus bacteriophage, and HU from T. maritima by systematically modifying charged surface residues and measuring binding constants and their salt-dependence. This program focuses on integrating research and teaching by offering the opportunity for both graduate and undergraduate students to acquire research experience and proficiency in techniques associated with analysis of protein-nucleic acid interactions. Graduate students will be integral to developing these research projects, and minority undergraduate students will continue to contribute to related projects.