HUMAN DNA POLYMERASE

人类DNA聚合酶

• 批准号: 8168576
• 负责人: Tahir H Tahirov
• 金额: $ 0.65万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168576
• 关键词: Active Sites; Amino Acids; Area; Biochemical; Complex; Computer Retrieval of Information on Scientific Projects Database; DBL Oncoprotein; DNA; DNA biosynthesis; Data; Funding; Genetic Recombination; Genomic Instability; Goals; Grant; Human; In Vitro; Individual; Institution; Lesion; Maps; Mediating; Molecular; Mutation; N-terminal; Phosphodiesterase I; Play; Polymerase; Positioning Attribute; Regulation; Reporting; Research; Research Personnel; Resources; Roentgen Rays; Role; Sampling; Source; Structure; TNFRSF5 gene; Testing; Two-Hybrid System Techniques; United States National Institutes of Health; X-Ray Crystallography; Yeasts; base; electron density; reconstitution; research study; scaffold; tumorigenesis

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human Pol ? is composed of four subunits and its activity is enhanced dramatically by interaction with PCNA. The largest p125 subunit has both polymerase and 3? to 5? exonuclease activities and mutations at the p125 active sites increase genomic instability and accelerate tumorigenesis.[8,9] The remaining three subunits, p50, p68, and p12 are thought to play a regulatory role, stimulate the polymerase activity of p125 by mediating additional interactions with PCNA, and stabilize the entire Pol ? complex. p50 serves as a scaffold for the assembly of Pol ? by interacting simultaneously with all of the other three subunits. Interestingly, many essential functions of Pol ?, including the regulation of replication, trans-lesion DNA synthesis and break-induced recombination, are mediated by the third subunit. Recently we reported the crystal structure of the complex between the second p50 subunit and the 144 amino acids N-terminal domain of the third p66 subunit (p66N) of human Pol ?. However, interactions of p50/p66 with p125 and p12 subunits, as well as DNA, remain unknown. We also succeeded in prepared of soluble monodisperse samples of p125, p50/p66, and p12, and confirmed the in vitro reconstitution of four-subunit Pol ?. Our long-term goal is to reveal the structure-based molecular mechanisms of human Pol ? function. Our short-term goal is to reveal the three-dimensional organization of Pol ? complex and locate the exact position of each subunit and map the areas involved in intersubunit interactions. To reach this goal we will combine cryo EM experiments with X-ray crystallography of individual domains and confirm our conclusions with biochemical and functional experiments. The data will allow as the positioning of the recently solved X-ray structure of human p50/p66 into the electron density maps obtained by Cryo EM, thus giving more complete information about the intersubunit interactions within Pol ?. We will test the defined subunit interactions with mutations and yeast two-hybrid assay.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 人类波尔？由四个亚基组成，其活性通过与 PCNA 的相互作用而显着增强。 最大的 p125 亚基同时具有聚合酶和 3?到5？ p125 活性位点的核酸外切酶活性和突变会增加基因组不稳定性并加速肿瘤发生。 [8,9] 其余三个亚基 p50、p68 和 p12 被认为发挥调节作用，通过介导与 PCNA 的额外相互作用来刺激 p125 的聚合酶活性，并稳定整个 Pol ?复杂的。 p50 充当 Pol 组装的支架？通过与所有其他三个亚基同时相互作用。 有趣的是，Pol α 的许多重要功能，包括复制调节、跨损伤 DNA 合成和断裂诱导重组，都是由第三个亚基介导的。 最近，我们报道了人Polα的第二个p50亚基和第三个p66亚基（p66N）的144个氨基酸N端结构域之间的复合物的晶体结构。然而，p50/p66 与 p125 和 p12 亚基以及 DNA 的相互作用仍然未知。 我们还成功制备了p125、p50/p66和p12的可溶性单分散样品，并证实了四亚基Polα的体外重构。 我们的长期目标是揭示人类 Pol 的基于结构的分子机制。功能。我们的短期目标是揭示 Pol 的三维组织？复杂并定位每个亚基的确切位置并绘制涉及亚基间相互作用的区域。为了实现这一目标，我们将冷冻电镜实验与各个域的 X 射线晶体学相结合，并通过生化和功能实验证实我们的结论。这些数据将允许将最近解析的人类 p50/p66 的 X 射线结构定位到冷冻电镜获得的电子密度图中，从而提供有关 Pol 内亚基间相互作用的更完整的信息。我们将测试定义的亚基与突变和酵母双杂交测定的相互作用。