STRUCTURAL STUDIES OF DNA REPLICATION AND REPAIR

DNA 复制和修复的结构研究

• 批准号: 6386308
• 负责人: GABRIEL WAKSMAN
• 金额: $ 27.29万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386308
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; Saccharomyces cerevisiae; X ray crystallography; adenosine triphosphate; bacterial proteins; endonuclease; enzyme activity; enzyme structure; enzyme substrate complex; fungal proteins; helicase; nucleic acid sequence; polymerization; protein engineering; protein structure function; site directed mutagenesis

The molecular structures of proteins involved in two aspects of DNA replication and repair, DNA unwinding and DNA polymerization, will be studied utilizing the techniques of x-ray crystallography. DNA bound and free forms of the proteins, as well as site-directed mutants, will be investigated. During the last grant period, four protein systems involved in these processes were studied: the DNA polymerase I from Thermus aquaticus, Escherichia coli single strand DNA-binding protein SSB, and the E.coli dimeric DNA-helicases Rep and UvrD. These proteins will be studied further, while the structural studies of one additional protein system, the DNA polymerase delta holoenzyme from Saccharomyces cerevisiae, will be initiated. The Taq DNA polymerase is involved in DNA repair, while the Polymerase delta holenzyme is the major replicative enzyme in eukaryotes. The Taq DNA polymerase is also an important biotechnological tool, used extensively in DNA sequencing and in the polymerase chain reaction. The crystal structures of nine different ligated states of an active N-terminal deletion of Taq polymerase corresponding to the Klenow fragment of E. coli of DNA polymerization have been determined. Additional complexes will be studied, as well as the full-length Taq polymerase which contains at its N-terminus a 5'-3' exonuclease activity. Studies of components of the polymerase delta holoenzyme will be initiated, which will shed light into the process of replicative DNA polymerization, which has been conserved from phage T4 to human. DNA unwinding is a fundamental process in DNA replication and repair. Several human diseases, including xeroderma pigmentosum and Cockayne's syndrome, involve defects in proteins that possess helicase activity. E. coli Rep and UvrD are involved in active unwinding of DNA, while E. coli SSB is involved in passive DNA helix destabilization. Rep and UvrD both function as dimmers and are motor proteins which use the energy derived from ATP-binding and hydrolysis to cycle kinetically through conformational states. The structures of the Rep helicase bound to a single-stranded DNA, as well as the structure of the DNA binding domain of E. coli SSB were determined. Structures of UvrD, as well as of complexes of SSB with DNA, are in the process of being solved. Various functionally-relevant complexes of these proteins, kinetically trapped at various stages of unwinding, will be studied. Such structures will provide information on the mechanism of active unwinding by DNA-helicases and of passive unwinding by helix-destabilizing SSB proteins.

蛋白质的分子结构涉及DNA的两个方面 复制和修复、DNA 解旋和 DNA 聚合，将 利用X射线晶体学技术进行研究。 DNA 结合和游离 将研究蛋白质的形式以及定点突变体。 在最后的资助期内，四个蛋白质系统参与了这些过程 研究人员：来自水生栖热菌、大肠杆菌的 DNA 聚合酶 I 单链 DNA 结合蛋白 SSB 和大肠杆菌二聚体 DNA 解旋酶 Rep 和UvrD。这些蛋白质将被进一步研究，而结构研究 另一种蛋白质系统，DNA聚合酶δ全酶 酿酒酵母将被启动。 Taq DNA 聚合酶参与 DNA 修复，而聚合酶 delta 孔酶是真核生物中主要的复制酶。 Taq DNA 聚合酶 也是一种重要的生物技术工具，广泛用于 DNA 测序 以及聚合酶链式反应。九种不同晶体结构 Taq聚合酶活性N端缺失的连接状态对应 大肠杆菌的Klenow片段的DNA聚合已经确定。 将研究其他复合物以及全长 Taq 聚合酶 其 N 末端含有 5'-3' 核酸外切酶活性。研究 聚合酶δ全酶的成分将被启动，这将 揭示了复制DNA聚合的过程，这一过程已被 从噬菌体 T4 到人类都是保守的。 DNA 解旋是 DNA 复制和修复的基本过程。一些 人类疾病，包括色素性干皮病和科凯恩综合症， 涉及具有解旋酶活性的蛋白质的缺陷。大肠杆菌代表和 UvrD 参与 DNA 的主动解旋，而大肠杆菌 SSB 参与 被动 DNA 螺旋不稳定。 Rep 和 UvrD 均起到调光器的作用 是利用 ATP 结合和水解产生的能量的运动蛋白 通过构象状态进行动力学循环。代表的结构 与单链 DNA 结合的解旋酶以及 DNA 的结构 确定了大肠杆菌SSB的结合域。 UvrD 的结构，以及 SSB 与 DNA 的复合物问题正在解决中。各种各样的 这些蛋白质的功能相关复合物，在动力学上被捕获 将研究放松的各个阶段。此类结构将提供 有关 DNA 螺旋酶主动解旋机制的信息以及 通过螺旋不稳定的 SSB 蛋白进行被动解旋。