Investigation of strand-specific DNA replication stress response mechanisms.

项目摘要 基因组不断地暴露于化学试剂中，这些化学试剂修改DNA碱基并破坏DNA。 DNA结构。这些未修复的DNA损伤可能成为复制过程中的障碍。这导致 阻止新生DNA合成并产生复制应激。因为父母的每一条DNA 通过不同的机制复制（前导链与滞后链合成）， 会产生独特的复制压力以前的研究使用 细胞培养和简单的体外复制系统已经描述了跨损伤合成，再引发， 和叉逆转作为途径，其功能是减轻复制应激。然而，这些方法 不能选择性地阻止前导链或滞后链的合成，也不能区分细胞机制 在每一条链上发挥作用。我假设前导链和滞后链的复制压力 通过不同的机制。我将用非洲爪蟾卵来区分链特异性机制 提取模型复制系统。我已经成功地构建了质粒底物， 前导和滞后链特异性病变。我的初步数据表明， 链对新生DNA合成的动力学有不同的影响，这表明不同的形式 复制压力的产生。这项建议将研究功能途径，缓解 链特异性复制应力，并评估每个途径的结果。在目标1中，我将使用凝胶- 基于分析，以监测形成的DNA中间体，并确定每个病变 最终克服了束缚。我还将使用定量蛋白质组学来监测 在复制前导链或滞后链病变期间招募或丢失。总之，这些方法将 确定在不同链上起作用的候选途径。在目标2中，我将使用功能丧失 一种确定链特异性途径如何控制并监测有害复制的方法 当选择的途径被灭活时的结果。这将通过选择性抑制来完成， 从细胞提取物中免疫耗竭候选蛋白。这项建议的结果将确定 对特定形式的复制应激作出反应并决定如何 采用各种机制来忠实地复制基因组。

Project Summary The genome is continuously exposed to chemical agents which modify DNA bases and damage the DNA structure. These unrepaired DNA lesions can become obstacles during replication. This results in stalling of nascent DNA synthesis and generates replication stress. Since each strand of parental DNA is replicated by distinct mechanisms (leading vs. lagging strand synthesis), DNA lesions encountered on different parental strands will generate unique forms of replication stress. Previous studies using cell culture and simple in vitro replication systems have described translesion synthesis, re-priming, and fork reversal as pathways which function to relieve replication stress. However, these approaches cannot selectively stall leading or lagging strand synthesis nor differentiate the cellular mechanisms that function on each strand. I hypothesize that leading and lagging strand replication stress are relieved by distinct mechanisms. I will differentiate between strand-specific mechanisms using the Xenopus egg extract model replication system. I have successfully constructed plasmid substrates which contain leading and lagging strand-specific lesions. My preliminary data demonstrate that lesions on different strands have different effects on the kinetics of nascent DNA synthesis, suggesting that different forms of replication stress are generated. This proposal will investigate the functional pathways which relieve strand-specific replication stress and evaluate the outcomes of each pathway. In Aim 1, I will use gel- based analyses to monitor the DNA intermediates that are formed and determine how lesions on each strand are eventually overcome. I will also use quantitative proteomics to monitor the proteins that are recruited or lost during replication of leading or lagging strand lesions. Together, these approaches will identify candidate pathways which function on different strands. In Aim 2, I will use a loss-of-function approach to determine how strand-specific pathways are controlled and monitor detrimental replication outcomes when select pathways are inactivated. This will be done by selective inhibition and immunodepletion of candidate proteins from the cell extracts. The findings of this proposal will define the specific cellular pathways which respond to specific forms of replication stress and determine how various mechanisms are employed to faithfully replicate the genome.