UMUDC/DNA POLYMERASE III INTERACTION

这项研究的长期目标是了解 大肠杆菌umuDC基因产物在大肠癌分子机制中的作用 诱导的诱变，并在细胞周期的控制，以响应 DNA损伤。 UmuD和UmuC已被证明允许DNA聚合酶III 以进行导致突变的跨损伤合成（44）。 最近来自步行者实验室的结果表明，完整的UmuD (the在跨损伤合成中无活性）与UmuC一起 参与响应DNA的细胞周期检查点机制 损伤（Opperman，T.，S. Murli和G. C.步行者，手稿在 制备）。步行者实验室的初步结果表明 UmuD和UmuD在物理上与β和β相互作用 DNA聚合酶III的亚基。 本提案中概述的实验 旨在深入了解UmuD，UmuD 和UmuC与DNA聚合酶III的这两个亚基，以及与其他 细胞蛋白质与它们在SOS突变和细胞中的作用有关 周期检查点控制。 我会从生物化学的角度分析蛋白质- 蛋白质相互作用，使用各种技术，包括凝胶 过滤和亲和层析，免疫共沉淀， 化学交联和表面等离子体共振（BIAcore）。 接下来的实验将集中在获得生物化学 UmuD和UmuC检查点控制的体外证据。

The long term goal of this research is to understand the role of the umuDC gene products of Escherichia coli in the molecular mechanism of induced mutagenesis and in the control of the cell cycle in response to DNA damage. UmuD and UmuC have been shown to permit DNA polymerase III to carry out translesion synthesis which results in mutations (44). Recent results from the Walker laboratory have shown that intact UmuD (the form inactive in translesion synthesis) together with UmuC participate in a cell cycle checkpoint mechanism in response to DNA damage (Opperman, T., S. Murli, and G. C. Walker, manuscript in preparation). Preliminary results from the Walker laboratory suggest that UmuD and UmuD interact physically with the epsilon and beta subunits of DNA polymerase III. Experiments outlined in this proposal are designed to provide insights into how the interaction of UmuD, UmuD and UmuC with these two subunits of DNA polymerase III, and with other cellular proteins is related to their roles in SOS mutagenesis and cell cycle checkpoint control. I will biochemically characterize protein- protein interactions using a variety of techniques including gel filtration and affinity chromatography, a co-immunoprecipitation, chemical cross-linking, and surface plasmon resonance (BIAcore). Subsequent experiments will concentrate on obtaining biochemical evidence of the UmuD and UmuC checkpoint control in vitro.