REPLICATION OF SIMPLE DNA REPEATS

简介(摘自申请者摘要)：本建议书重点介绍 体内复制简单DNA重复序列的机制。长度的变化 会导致许多人类神经系统疾病， 某些形式的癌症。人们认为，这些变化是由异常引起的 重复区域的复制，但支持这一概念的直接数据 是稀有的。使用复制中间产物的电泳分析作为 工具和细菌质粒作为模型系统，首席研究员 通过几个步骤直接显示了复制分叉进程 重复的DNA伸展异常缓慢，而落后的DNA链 重复通常是复制不足的。他发现有三种不同的机制 解释了不同重复导致的复制停滞。一种机制 可能是一个稳定的二级结构的重复DNA位于 滞后的链模板。另一个原因是转录停滞在 DNA重复拉伸，然后复制受阻。最后，合作 与第三组重复序列结合的蛋白质负责复制 衰减。首席研究人员假设复制停滞 可能是重复长度不稳定的原因。在这份提案中，他 将集中于这些机制在酵母菌和 细菌系统。具体来说，他将研究三核苷酸的复制 重复序列在酵母实验系统中的应用 复制中间体。使用基因替换方法，他将获得 酵母复制装置的不同突变体，以确定 通过DNA重复影响复制分叉进程的组件。这个 然后首席调查员将研究变化之间的相关性 三核苷酸重复序列在酵母中的复制及其扩增率 复制型突变体。他还将研究转录和 大肠杆菌中高嘌呤-高嘧啶重复序列中的复制停滞 和酵母系统。他将决定STESTED的精细结构 用突变分析和化学方法研究体内和体外的中间体 脚印。最后，他将研究复制分叉的机制 蛋白质与重复的DNA延伸相结合而引起的衰减。为此， 他将提纯和鉴定结合在一起的新型大肠杆菌蛋白 具体到d(G-A)n d(T-C)n个重复。我们的长期目标是理解 复制简单DNA重复序列在它们的维持和恢复中所起的作用 长度多态。

DESCRIPTION (adapted from applicant's abstract): This proposal focuses on the mechanisms of replication of simple DNA repeats in vivo. Changes in the length of such repeats are responsible for numerous human neurological diseases and some forms of cancer. It is believed that these changes are caused by abnormal replication of the repetitive tracts, but direct data supporting this notion are scarce. Using electrophoretic analysis of replication intermediates as a tool and bacterial plasmids as a model system, the principal investigator has directly shown that progression of the replication fork through several repetitive DNA stretches is unusually slow and the lagging DNA strand of the repeat is often underreplicated. He has found that three different mechanisms account for the replication stalling caused by different repeats. One mechanism is likely to be a stable secondary structure of the repeated DNA situated on the lagging strand template. Another is due to transcription stalling at the repeated DNA stretch followed by replication blockage. Finally, cooperative protein binding to the third group of repeats is responsible for replication attenuation. The principal investigator hypothesizes that replication stalling could be responsible for the repeat-length instability. In this proposal, he will focus on the detailed characterization of these mechanisms in yeast and bacterial systems. Specifically, he will study the replication of trinucleotide repeats in a yeast experimental system using electrophoretic analysis of replication intermediates. Using gene replacement methods, he will obtain different mutants of the yeast replication apparatus in order to determine the components affecting the replication fork progression through DNA repeats. The principal investigator will then study the correlation between changes in the replication of trinucleotide repeats and their expansion rates in yeast replicative mutants. He will also study the relation between transcription and replication-stalling within homopurine-homopyrimidine repeats in the E. coli and yeast systems. He will determine the fine structure of stalled intermediates in vivo and in vitro using mutational analysis and chemical footprinting. Finally, he will study the mechanism of replication fork attenuation caused by protein binding to repetitive DNA stretches. To this end, he will purify and characterize the novel E. coli protein that binds specifically to d(G-A)n d(T-C)n repeats. The long-term goal is to understand the role played by replication of simple DNA repeats in their maintenance and length polymorphism.