STRUCTURAL STUDIES OF DNA REPLICATION AND REPAIR

描述：研究者建议研究分子结构 两种非序列特异性DNA结合蛋白参与DNA修复 和复制、Thermos aquaticus DNA聚合酶和E.大肠杆菌单 链DNA结合蛋白质。 免费和DNA- 将研究蛋白质的结合形式，以及位点- 定向突变体 预计这些研究将提供有关以下方面的见解 DNA聚合和DNA解旋的机制。 Taq DNA聚合酶活性N端缺失的结构 对应于大肠杆菌DNA聚合酶的Klenow片段， 确定为2.5埃分辨率，并将被细化到更高 分辨率 进一步的研究将涉及与DNA的复合物， 脱氧核苷酸和双脱氧核苷酸以提供信息 DNA聚合的机制，并将形成基础， 试图改善酶。 大肠杆菌单链结合蛋白是DNA 复制，DNA修复过程，并在RecA介导的同源 重组 SSB的胰凝乳蛋白酶片段（SSBc），含有 ssDNA结合结构域已经在低分辨率下结晶和解析。 已经收集了一个2.5埃的原生数据集，可能是SSBc 并且正在努力确定高分辨率结构。 硒代蛋氨酸已被纳入SSB和硒代蛋氨酸 含有SSBc的晶体已经结晶。 晶体在同一个 空间群与具有相同晶胞的天然晶体相同。 闪- 研究出了这些晶体和同步加速器的冷却方法 布鲁克海文国家实验室的时间将允许收集 高分辨率多波长异常散射数据。 最近， 已经生长了相当大的DNA/SSBc复合物晶体。

DESCRIPTION: The investigator proposes to study the molecular structures of two non-sequence specific DNA-binding proteins involved in DNA repair and replication, Thermos aquaticus DNA polymerase and E. coli single strand DNA- binding protein using X-ray crystallography. Free and DNA- bound forms of the proteins will be investigated, as well as site- directed mutants. These studies are expected to provide insight into the mechanisms of DNA polymerization and DNA unwinding. The structure of an active N-terminal deletion of Taq DNA polymerase corresponding to the Klenow fragment of E coli DNA polymerase has been determined to 2.5 Angstroms resolution and will be refined to higher resolution. Additional studies will involve complexes with DNA, deoxynucleotides and dideoxynucleotides in order to provide information on the mechanism of DNA polymerization and will form the basis for attempts to improve the enzyme. The E coli single strand binding protein is essential for DNA replication, DNA repair processes, and in RecA-mediated homologous recombination. A chymotryptic fragment of SSB (SSBc) containing the ssDNA-binding domain has been crystallized and solved at low-resolution. A 2.5 Angstroms native data set, presumably of SSBc, has been collected and efforts are underway to determine a high resolution structure. Selenomethionine has been incorporated into SSB and selenomethionine containing SSBc has been crystallized. The crystals are in the same space group as the native crystals with the same unit cell. Flash- cooling methods have been worked out for these crystals and synchrotron time at the Brookhaven National Laboratory will allow the collection of high resolution multi-wavelength anomalous scattering data. Recently, sizable crystals of a DNA/SSBc complex have been grown.