权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

ENDONUCLEASE/TOPOISOMERASE NAEI

核酸内切酶/拓扑异构酶 NAEI

基本信息

批准号：
6490084
负责人：
MICHAEL D TOPAL
金额：
$ 25.25万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-01 至 2003-12-31
项目状态：
已结题

项目摘要

DNA topoisomerases, recombinases, endonucleases and ligases are basic to the genetic machinery of the cell. These enzymes are ubiquitous and essential for replication, transcription, recombination, and repair of DNA. Therefore, understanding their mechanisms of action is important for understanding the basis for genetic instability diseases such as cancer. Endonuclease NaeI appears to hold the key that relates these DNA processing enzymes to each other. No sequence similarity is detectable between NaeI and the topoisomerases and recombinases. A stretch of 10 amino acids in NaeI, however, has significant similarity to the adenylation/joining active site of DNA ligase I. A single L43K amino acid change within this region of NaeI radically transforms NaeI activity from endonuclease to that of a DNA topoisomerase and recombinase. The biochemical basis for this altered activity is unknown and the mechanism of this novel topoisomerase remains to be elucidated. The discovery of topoisomerase and recombinase activity within NaeI endonuclease unites what were previously believed to be independently derived DNA processing activities. The specific aims of this proposal are to: (i) characterize the covalent attachment between NaeI/NaeI-43K and DNA; (ii) biochemically map the domain structures of NaeI and NaeI-L43K by limited proteolysis; (iii) solve the three-dimensional structure of NaeI protein complexed with a DNA fragment; (iv) improve the expression of NaeI-43K in vitro in order to isolate large amounts of this protein for crystallization studies, and (v) explore the structure-function relationships, implied by the crystal structures, by mutagenic and biochemical means.

DNA拓扑异构酶、重组酶、内切核酸酶和连接酶是是细胞遗传机制的基础这些酶普遍存在，并且对于复制，转录，重组和DNA修复。因此，了解他们的作用机制对于理解遗传不稳定性疾病，如癌症。核酸内切酶NaeI 似乎掌握着将这些DNA加工酶彼此之间。在NaeI之间未检测到序列相似性以及拓扑异构酶和重组酶。一段10氨基然而，NaeI中的酸与 DNA连接酶I的腺苷酸化/连接活性位点。一个L43 K NaeI这一区域内的氨基酸变化从根本上改变了从核酸内切酶到DNA拓扑异构酶的NaeI活性和重组酶。这种活动改变的生化基础这种新型拓扑异构酶的作用机制仍不清楚有待阐明。拓扑异构酶和重组酶的发现 NaeI内切核酸酶内的活性结合了以前被认为是独立衍生的DNA加工活动。本提案的具体目标是： NaeI/NaeI-43 K与DNA之间的共价连接;（ii）用生物化学方法绘制NaeI和NaeI-L43 K的结构域，有限的蛋白水解;（iii）解决三维结构与DNA片段复合的NaeI蛋白;（iv）提高在体外表达NaeI-43 K，以分离大量用于结晶研究，以及（v）探索晶体所暗示的结构-功能关系结构，通过诱变和生物化学手段。