INITIATION OF LAGGING-STRAND DNA SYNTHESIS

这个实验室的主要工作是为了 发展对复制机制如何获得 对DNA模板的访问并随后保持绑定 巨大的距离，同时积极地综合了领先的- 以及落后的DNA链。为此，进行了详细的调查 在复制叉处存在的酶的活性是 呈上了。特别关注蛋白质--蛋白质 和蛋白质-DNA相互作用，这些都是复制的特征 叉子。将继续进行研究，旨在描述 原始体的相互作用(一种移动的启动装置， 需要七种大肠杆菌编码的蛋白质进行组装，并且可以 在DNA上的特定位置组装(原始体组装位置)， 连续迁移5‘端至3’端，偶尔合成一条引物) 有原始体组装地点。这种蛋白质负责 原粒运动将被确立，这三个基因 未定义的原始体蛋白(复制因子Y(蛋白n‘)) 和蛋白质n和n“)将被分子克隆，它们的基因 特色化的。 复制叉子的结构和移动将是 使用支持滚动的新型底漆模板进行研究- 环状DNA复制其中合成的前导-和 滞后的DNA链连接在一起。蛋白质--蛋白质 DNA聚合酶III所需的相互作用和作用 在叉子上连接复制机械的全酶将 被建立起来。复制分叉移动的速率将为 可测量的复制进度和影响复制进度的因素 叉子将被识别出来。

A major part of the effort of this laboratory is directed at developing an understanding of how the replication machinery gains access to the DNA template and subsequently remains bound over tremendous distances while actively synthesizing both the leading- and lagging-strands of DNA. To this end, detailed investigations of the activities of enzymes present at the replication fork are presented. Particular attention is given to the protein-protein and protein-DNA interactions that characterize the replication fork. Studies will be continued that are designed to characterize the interaction of the primosome (a mobile priming apparatus that requires seven E. coli-encoded proteins for assembly and which can assemble at a specific site on the DNA (primosome assembly sites), migrate processively 5' to 3' and occasionally synthesize a primer) with primosome assembly sites. The protein responsible for primosome movement will be established and the three genetically undefined primosomal proteins (replication factor Y (protein n') and proteins n and n") will be molecularly cloned and their genes characterized. The structure and movement of the replication fork will be investigated using novel primer-templates that sustain rolling- circle DNA replication in which synthesis of the leading- and lagging-strands of DNA are coupled. The protein-protein interactions required and the role of the DNA polymerase III holoenzyme in coupling the replication machinery at the fork will be established. Rates of replication fork movement will be measured and factors that influence progress of the replication fork will be identified.