HUMAN DNA POLYMERASE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，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相互作用而显著增强。 最大的p125亚基有聚合酶和3？到5？外切核酸酶活性和p125活性位点的突变增加了基因组的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的额外相互作用刺激p125的聚合酶活性，并稳定整个Pol？复杂. p50作为一个脚手架的组装波尔？通过同时与其他三个亚单位相互作用。 有趣的是，Pol？的许多基本功能，包括复制的调节、跨损伤DNA合成和断裂诱导的重组，都是由第三亚基介导的。 最近我们报道了人Pol？第二p50亚基与第三p66亚基N端144个氨基酸组成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。 我们还成功地制备了p125、p50/p66和p12的可溶性单分散样品，并证实了四亚基Pol？的体外重建。 我们的长期目标是揭示人类Pol？功能我们的短期目标是揭示波尔？复合物，并定位每个亚基的确切位置，并绘制亚基间相互作用所涉及的区域。为了达到这个目标，我们将结合联合收割机冷冻EM实验与X射线晶体学的个别领域，并确认我们的结论与生化和功能实验。这些数据将允许将最近解决的人类p50/p66的X射线结构定位到通过Cryo EM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信息。我们将测试定义的亚基与突变的相互作用和酵母双杂交试验。

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.