HUMAN DNA POLYMERASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人形波兰人？由四个亚基组成，其活性通过与增殖细胞核抗原的相互作用而显著增强。最大的p125亚基既有聚合酶又有3？到5点？核酸外切酶的活性和p125活性部位的突变增加了基因组的不稳定性，加速了肿瘤的发生。[8，9]剩下的三个亚单位p50、p68和p12被认为起着调节作用，通过介导与增殖细胞核抗原的额外相互作用来刺激p125的聚合酶活性，并稳定整个POL？很复杂。P50作为组装POL？的支架通过同时与所有其他三个亚基相互作用。有趣的是，POL？的许多基本功能，包括复制调控、跨损伤DNA合成和断裂诱导的重组，都是由第三亚单位介导的。 最近，我们报道了人Pol？的第二个p50亚基与第三个p66亚基(P66N)的144个氨基酸N-末端结构域之间的复合体的晶体结构。然而，p50/p66与p125和p12亚基以及DNA之间的相互作用仍不清楚。我们还成功地制备了p125、p50/p66和p12的可溶单分散样品，证实了四个亚基Pol？的体外重组。 我们的长期目标是揭示基于结构的人类POL？分子机制。功能。我们的短期目标是揭示POL？的立体组织。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，我们将结合低温EM实验和X射线结晶学的个别结构域，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决的人类p50/p66的X射线结构定位到Cryo EM获得的电子密度图中，从而提供关于POL？亚基间相互作用的更完整的信息。我们将用突变和酵母双杂交试验来测试确定的亚基相互作用。

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.