UMUDC/DNA POLYMERASE III INTERACTION

这项研究的长期目标是了解 大肠埃希菌umuDC基因产物在分子机制中的作用 诱导突变和对细胞周期的控制 DNA损伤。UMUD和UMUC已被证明允许DNA聚合酶III 进行跨病变合成，导致突变(44)。 沃克实验室的最新结果表明，完整的UmuD (在跨病变合成中不活跃的形式)与UMUC一起 参与细胞周期检查点机制以响应DNA 损害(Opperman，T.，S.Murli和G.C.Walker，手稿in 准备)。沃克实验室的初步结果表明 UmuD和UmuD与epsilon和beta相互作用 DNA聚合酶亚基III.本提案中概述的实验 旨在深入了解UmuD、UmuD和UmuD之间的相互作用 和UMUC与DNA聚合酶III的这两个亚单位，以及与其他 细胞蛋白与其在SOS诱变和细胞中的作用 循环检查点控制。我将对蛋白质进行生化表征- 使用包括凝胶在内的各种技术进行蛋白质相互作用 过滤和亲和层析，一种免疫共沉淀， 化学交联和表面等离子体共振(Biacore)。 随后的实验将集中在获得生化 UMUD和UMUC检查站体外控制的证据。

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.