Examining the Role of Exo V in Repairing DNA Damage

检查 Exo V 在修复 DNA 损伤中的作用

• 批准号: 6829440
• 负责人: ANDRES A LARREA
• 金额: $ 3.2万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829440
• 关键词: DNA damage; DNA repair; SDS polyacrylamide gel electrophoresis; X ray crystallography; affinity chromatography; affinity labeling; bacterial genetics; enzyme activity; exodeoxyribonuclease; exonuclease; genetic recombination; plasmids; predoctoral investigator; protein protein interaction; protein purification; protein structure function; yeast two hybrid system

DESCRIPTION (provided by applicant): Life depends upon the faithful transmission of genetic information from one generation to the next. Unfortunately DNA damage occurs and, when it does, it can lead to the loss of important genetic information. My work focuses on understanding how two different groups of bacteria have chosen to solve the problem presented by one very harmful type of DNA damage: double-strand breaks. The first part of this proposal focuses on RecBCD, which is a heterotrimeric protein from E. coli. RecBCD is a potent double strand 5'-3' and 3'-5' nuclease, helicase, and ATPase. It is highly processive, digesting kilobases of DNA per binding event. It is, however, also a recombinase that promotes homologous recombination. These two paradoxical roles are mediated by the 8-nucleotide chi sequence (5'-GCTGGTGG-3'). The second part of the proposal focuses on AddAB, the two-subunit enzyme responsible for Exo V activity in T. tengcongensis and other gram-positive bacteria. AddAB has the same activity and role in the cell as RecBCD except it responds to a different chi sequence. In this project I will use a variety of techniques including crystallography, genetics, and enzymology to determine the structure and dissect the regulation of these processive nucleases.

描述（由申请人提供）：生命取决于遗传信息从一代到下一代的忠实传递。不幸的是，DNA 损伤会发生，一旦发生，可能会导致重要遗传信息的丢失。我的工作重点是了解两组不同的细菌如何选择解决一种非常有害的 DNA 损伤类型（双链断裂）所带来的问题。该提案的第一部分重点关注 RecBCD，它是来自大肠杆菌的异三聚体蛋白。 RecBCD 是一种有效的双链 5'-3' 和 3'-5' 核酸酶、解旋酶和 ATP 酶。它具有高度持续性，每次结合事件都会消化数千碱基的 DNA。然而，它也是一种促进同源重组的重组酶。这两种矛盾的作用是由 8 核苷酸 chi 序列 (5'-GCTGGTGG-3') 介导的。该提案的第二部分重点关注 AddAB，这是一种负责 T. tengcongensis 和其他革兰氏阳性细菌中 Exo V 活性的双亚基酶。 AddAB 在细胞中具有与 RecBCD 相同的活性和作用，只是它响应不同的 chi 序列。在这个项目中，我将使用包括晶体学、遗传学和酶学在内的多种技术来确定这些进行性核酸酶的结构并剖析其调控。