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.

蛋白质的分子结构参与DNA的两个方面 复制和修复，DNA解开和DNA聚合将是 利用X射线晶体学技术进行了研究。 DNA结合和自由 将研究蛋白质的形式以及定位的突变体。 在上一个赠款期间，这些过程中涉及四个蛋白质系统 研究了：来自Thermus Aquaticus，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-壳酶REP 和uvrd。这些蛋白质将进一步研究，而结构研究 在另一种蛋白质系统中，DNA聚合酶三角酶的全酶 酿酒酵母将开始。 TAQ DNA聚合酶参与DNA修复，而聚合酶三角洲 holenzyme是真核生物中的主要复制酶。 TAQ DNA聚合酶 也是重要的生物技术工具，广泛用于DNA测序 并在聚合酶链反应中。九种不同的晶体结构 TAQ聚合酶的活性N末端缺失的连接状态相应 已经确定了DNA聚合大肠杆菌的Klenow片段。 将研究其他复合物以及全长TAQ聚合酶 它在其N末端包含5'-3'核酸外切酶活性。研究 将启动聚合酶三角酶的成分，这将 将光浸入复制性DNA聚合过程中， 从噬菌体T4到人保守。 DNA放弃是DNA复制和修复的基本过程。一些 人类疾病，包括静脉皮色素和Cockayne的综合症， 涉及具有解旋酶活性的蛋白质缺陷。大肠杆菌代表和 UVRD参与DNA的积极放松，而大肠杆菌SSB参与 被动DNA螺旋不稳定。 rep和uvrd都充当调光器， 是使用源自ATP结合和水解的能量的运动蛋白 通过构象状态进行运动循环。代表的结构 与单链DNA结合的解旋酶以及DNA的结构 确定大肠杆菌SSB的结合结构域。 UVRD的结构以及 SSB与DNA的复合物正在解决过程中。各种各样的 这些蛋白质与功能相关的复合物，动力学捕获在 将研究各个阶段的放松阶段。这样的结构将提供 关于通过DNA螺旋酶和dna螺旋酶固定的机制的信息 被螺旋可固定的SSB蛋白通过被动放松。