Examining the Role of Exo V in Repairing DNA Damage

描述(由申请者提供)：生命依赖于遗传信息的忠实代代相传。不幸的是，DNA损伤会发生，当它发生时，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是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。这项提案的第一部分集中在RecBCD，这是一种来自大肠杆菌的异源三聚体蛋白。RecBCD是一种有效的双链5‘-3’和3‘-5’核酸酶、解旋酶和ATPase。它是高度进行性的，每次结合事件都能消化几千个碱基的DNA。然而，它也是一种促进同源重组的重组酶。这两个自相矛盾的作用是由8核苷酸chi序列(5‘-GCTGGTGG-3’)介导的。提案的第二部分侧重于AddAB，这是一种两亚单位酶，负责T.tengcongensis和其他革兰氏阳性细菌的Exo V活性。AddAB在细胞中具有与RecBCD相同的活性和作用，只是它对不同的CHI序列做出反应。在这个项目中，我将使用包括结晶学、遗传学和酶学在内的各种技术来确定这些过程核酸酶的结构并剖析其调节机制。

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.