INHIBITORS OF SITE-SPECIFIC RECOMBINATION

位点特异性重组抑制剂

• 批准号: 3282957
• 负责人: Michael Fennewald
• 金额: $ 12.77万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282957
• 关键词: DNA; DNA binding protein; DNA directed DNA polymerase; DNA topoisomerases; Escherichia coli k12; adenosinetriphosphatase; autoradiography; bacterial genetics; bacteriophage lambda; chemical binding; chromosome deletion; deoxyribonuclease I; enzyme inhibitors; gel electrophoresis; gene deletion mutation; gene expression; genetic manipulation; genetic recombination; genetic regulation; neocarzinostatin; nucleic acid sequence; plasmids; point mutation; radiotracer; transposon /insertion element

The objective of this research is to understand the biochemical mechanism by which the Tn3 transposon causes insertions and other genetic rearrangements. The immediate goal is to characterize the activities and mechanisms of the Tn3 resolvase because these are the two Tn3-encoded proteins needed for transposition. We have found that the transposase is a site- specific DNA binding protein that binds to the inverted repeats at the ends of Tn3. We will determine the extent of binding by transposase to the inverted repeats down to the nucleotide level, and will also test altered inverted repeats. Transposase also has a DNA-dependent ATPase and we will optimize the conditions for this activity and search for other activities expected for a transposase protein. Establishment of an in vitro system for transposition will also be attempted. For resolvase, the mode of action of a series of chemical inhibitors will be investigated. Several of these inhibitors do not block DNA binding but do block DNA cleavage and recombination and we will test if they block the formation or maturation of a synaptic complex. We will also test the effect of these inhibitors for the gin inversion system in vitro. Inhibitors of DNA binding by resolvase will be testing for their effect on proteolytic fragments of resolvase. Along with isolation of resistant mutants, this should allow the identification of the domains of resolvase involved in DNA binding.

这项研究的目的是了解生化 TN3转座子引起插入和 其他遗传重排。 直接目标是 表征TN3分解的活动和机制 因为这些是两个TN3编码的蛋白 换位。 我们发现转座酶是一个位点 - 特定的DNA结合蛋白与在 TN3的末端。 我们将确定绑定的程度 转座酶至倒的倒置重复至核苷酸水平， 并将测试变更的倒重复序列。 转座酶也有 DNA依赖性ATPase，我们将优化 这项活动并搜索预期的其他活动 转座酶蛋白。 建立一个体外系统 也将尝试转置。 对于分解， 将研究一系列化学抑制剂的作用。 这些抑制剂中的几种不阻止DNA结合，但会阻塞 DNA裂解和重组，我们将测试它们是否阻止 突触复合物的形成或成熟。 我们也会 测试这些抑制剂对杜松子酒反转系统的影响 体外。 通过分解的DNA结合的抑制剂将测试 它们对分解的蛋白水解片段的影响。 以及 抗性突变体的隔离，这应该允许识别 参与DNA结合的分解区域的结构域。