STRUCTURAL STUDIES OF DNA REPLICATION AND REPAIR

涉及DNA两个方面的蛋白质的分子结构 复制和修复，DNA解离和DNA聚合，将是 利用X射线结晶学技术进行了研究。DNA绑定和自由 这些蛋白质的形式以及定点突变体将被研究。 在上一次赠款期间，有四个蛋白质系统参与了这些过程 的DNA聚合酶I进行了研究。 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 在一个额外的蛋白质系统中，来自 酿酒酵母，将被启动。 Taq DNA聚合酶参与DNA修复，而聚合酶Delta 全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 在聚合酶链式反应中。九种不同化合物的晶体结构 相应的Taq聚合酶活性N端缺失的连接状态 对DNA聚合的大肠杆菌Klenow片段进行了测定。 还将研究更多的复合体以及全长Taq聚合酶 其N端含有5‘-3’核酸外切酶活性。的研究 聚合酶三角洲全酶的组成部分将被启动，这将 揭示了复制DNA聚合的过程，这已经被 从噬菌体T4到人类的保守性。 DNA解离是DNA复制和修复的基本过程。几个 人类疾病，包括着色性干皮病和Cockayne综合征， 涉及具有解旋酶活性的蛋白质中的缺陷。大肠埃希菌代表和 UvrD参与DNA的主动解离，而E.coliSSB参与 被动的DNA螺旋不稳定。REP和UVRD都用作调光器和 是使用来自ATP结合和水解的能量的马达蛋白吗？ 在构象状态中动态循环。代表的结构 与单链DNA结合的解旋酶，以及DNA的结构 确定了E.ColiSSB的结合区。Uvrd的结构，以及 关于SSB与DNA的复合体，正在解决的过程中。五花八门 这些蛋白质的功能相关的复合体，动力学地捕获在 平仓的各个阶段，都将被研究。这样的结构将提供 DNA解旋酶和DNA解旋酶活性解离机制的研究进展 螺旋破坏SSB蛋白的被动解离。

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.