STRUCTURAL STUDIES OF DNA REPLICATION AND REPAIR

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， 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。 在上一次资助期间，参与这些过程的四种蛋白质系统 研究了水生栖热菌、大肠杆菌、 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 另一个蛋白质系统，DNA聚合酶δ全酶， 酿酒酵母，将启动。 Taq DNA聚合酶参与DNA修复，而聚合酶δ Holenzyme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 和聚合酶链反应中。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对应于 与E.大肠杆菌的DNA聚合。 将研究其他复合物，以及全长Taq聚合酶 其在其N-末端含有5 ′-3 ′核酸外切酶活性。研究 聚合酶δ全酶的组分将被启动， 揭示了复制DNA聚合的过程， 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。几 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。E. coli Rep和 UvrD参与DNA主动解旋，而E. coli SSB参与了 被动DNA螺旋不稳定。Rep和UvrD都用作调光器， 是利用ATP结合和水解产生的能量的马达蛋白 在构象状态中进行动力学循环。代表的结构 解旋酶与单链DNA的结合，以及DNA的结构 E. coli SSB的表达。UvrD的结构，以及 SSB与DNA的复合物，正在解决的过程中。各种 这些蛋白质的功能相关复合物，动力学捕获在 将研究解旋的各个阶段。这些结构将提供 关于DNA解旋酶主动解旋机制和 被动解旋螺旋不稳定SSB蛋白。

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.