ENDONUCLEASE/TOPOISOMERASE NAEI

核酸内切酶/拓扑异构酶 NAEI

• 批准号: 6342904
• 负责人: MICHAEL D TOPAL
• 金额: $ 24.52万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342904
• 关键词: DNA; DNA topoisomerases; SDS polyacrylamide gel electrophoresis; X ray crystallography; active sites; chimeric proteins; crystallization; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; point mutation; protein sequence; restriction endonucleases; site directed mutagenesis; stoichiometry; structural biology; western blottings

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、核酸内切酶和连接酶