REPLICATION OF SIMPLE DNA REPEATS

描述（改编自申请人的摘要）：本提案侧重于 简单DNA重复序列在体内的复制机制。长度的变化 这种重复序列的一部分是许多人类神经疾病的原因， 某些形式的癌症。据认为，这些变化是由异常 复制的重复路段，但直接数据支持这一概念 是稀缺的使用复制中间体的电泳分析作为 工具和细菌质粒作为模型系统，主要研究者 直接显示了复制分叉通过几个 重复的DNA延伸异常缓慢， repeat重复is often经常underreplicated复制.他发现有三种不同的机制 考虑到由不同的重复引起的复制停止。一种机制 很可能是重复DNA的稳定二级结构， 滞后链模板。另一个是由于转录停滞在 重复的DNA拉伸，然后是复制阻断。最后，合作 与第三组重复序列结合的蛋白质负责复制 衰减首席研究员假设复制停滞 可能是造成重复长度不稳定的原因。在这份提案中，他 将集中在这些机制在酵母的详细特征， 细菌系统具体来说，他将研究三核苷酸的复制， 重复在酵母实验系统中使用电泳分析， 复制中间体通过基因置换的方法，他将获得 酵母复制装置的不同突变体，以确定 通过DNA重复序列影响复制叉进程的组分。的 然后，首席研究员将研究 三核苷酸重复序列在酵母中的复制及其扩增速率 复制突变体他还将研究转录与 在E.杆菌 和酵母系统。他将确定失速的精细结构 使用突变分析和化学分析在体内和体外研究中间体 脚印最后，他将研究复制叉的机制 由蛋白质与重复DNA片段结合引起的衰减。为此目的， 他将净化和刻画小说E。大肠杆菌蛋白质， 特别是d（G-A）n d（T-C）n重复。长期目标是了解 简单DNA重复序列的复制在其维持中所起的作用， 长度多态性

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.