BYPASS MECHANISMS IN EUKARYOTIC REPLICATION

项目总结/摘要 染色体由一种称为复制体的复杂全酶复制。障碍是例行谈判 通过复制体与确保基因组完整性的辅助机制，其变异可导致 染色体不稳定性和包括癌症在内的多种疾病。候选人的长期目标是 了解遗传和表观遗传保真度的分子基础，有可能改善治疗 和/或预防疾病。在这个提议中，候选人将使用一个功能齐全的复制体重组 从30多个纯多肽中研究复制体如何绕过基因组中经常发生的障碍 同时加强遗传和表观遗传的完整性。在第一个具体目标中， 目前的工作，病变旁路的分子机制的复制体将详细阐述，重点是 关于检查点激酶Mec 1和Rad 53以及Mrc 1/Tof 1/Csm 3（MTC）复合物如何改变 复制体，同时调节病变旁路。几个关键的复制体组分与 MTC复合物将通过微尺度热泳（MST）和交联质谱（XL-100）探测。 MS）。除了生化实验外，单分子方法将用于探索机制 MTC复合物对复制体的调节，在活性期间用荧光解析复制体组分， DNA复制单分子FRET实验将用于独立阶段，以探索如何 MTC影响复制体的结构动力学。在第二个目标中，候选人将调查 核小体旁路的复制体，集中在复制后的命运组蛋白在指导 相位使用荧光富集的组蛋白，复制偶联的组蛋白沉积将通过一种新的方法来跟踪。 第一次尝试在空间上解决领先与落后的链产品与光学组合 捕获荧光和流动沿着以珠子为基础的生化实验， 区分不同的表观遗传模型。在独立阶段， 将用MST和XL-MS测定复制体和FACT（一种组蛋白伴侣）的分子量。 各种分子伴侣的机制将使用单分子FRET实验监测 在真实的时间内染色质重塑的空间分布，也决定了组蛋白修饰在 重塑该项目的指导阶段将在Michael奥唐纳博士的实验室进行 （导师）和洛克菲勒大学（世界一流的研究环境）的刘世新博士（联合导师）。的 候选人提出的研究的成功关键取决于使用先进的整合单分子 技术，以及XL-MS。因此，候选人寻求与操纵和检测的密集培训， 在共同导师的国家的最先进的设施，除了XL-MS与布赖恩Chait博士和 李（合作者）。候选人还计划了一些活动，以改善指导，实验室管理， 科学沟通和专业技能，使成功过渡到一个独立的职业生涯。

PROJECT SUMMARY/ABSTRACT Chromosomes are copied by a complex holoenzyme called the replisome. Obstacles are routinely negotiated by the replisome with auxiliary mechanisms that ensure genomic integrity, aberrance of which can lead to chromosome instability and a broad range of diseases including cancer. The candidate’s long term goal is to understand the molecular basis for genetic and epigenetic fidelity, with the potential to improve the treatment and/or prevention of disease. In this proposal, the candidate will use a fully functional replisome reconstituted from over 30 pure polypeptides to study how replisomes bypass obstacles that regularly occur in the genome while enforcing genetic and epigenetic integrity across generations. In the first specific aim the candidate’s current work on the molecular mechanisms of lesion bypass by the replisome will be elaborated, with a focus on how checkpoint kinases Mec1 and Rad53 and the Mrc1/Tof1/Csm3 (MTC) complex modify the activity of the replisome while regulating lesion bypass. Interactions between several key replisome components and the MTC complex will be probed by microscale thermophoresis (MST) and cross-linking mass spectrometry (XL- MS). In addition to biochemical experiments, single-molecule approaches will be used to probe the mechanism of replisome regulation by the MTC complex, resolving replisome components with fluorescence during active replication on DNA. Single-molecule FRET experiments will be used in the independent phase to probe how MTC affects the structural dynamics of the replisome. In the second aim, the candidate will investigate nucleosome bypass by the replisome, focusing on the post-replication fate of histones during the mentored phase. Using histones enriched for fluorescence, replication-coupled histone deposition will be tracked by a first-of-its-kind attempt at spatially resolving leading vs. lagging strand products with a combination of optical trapping, fluorescence, and flow. Along with bead-based biochemical experiments, the results will help differentiate between models of epigenetic inheritance. In the independent phase, interactions between the replisome and FACT, a histone chaperone, will be determined with MST and XL-MS. The molecular mechanisms of various chaperones will be probed using single-molecule FRET experiments monitoring the spatiokinetics of chromatin remodeling in real time, also determining the role of histone modifications in remodeling. The mentored phase of the project will be conducted in the laboratories of Dr. Michael O’Donnell (mentor) and Dr. Shixin Liu (co-mentor) at Rockefeller University, a world-class research environment. The success of the candidate’s proposed research depends critically on using advanced integrative single-molecule techniques, as well as XL-MS. Thus, the candidate seeks intensive training with manipulation and detection of individual molecules in the co-mentor’s state-of-the-art facilities, in addition to XL-MS with Drs. Brian Chait and Yi Shi (collaborators). The candidate also has also planned activities to improve mentoring, lab management, scientific communication, and professional skills, enabling a successful transition to an independent career.