Bypass Mechanisms in Eukaryotic Replication

项目总结 染色体由一种名为复制体的复杂全酶复制。障碍是例行公事地谈判 通过带有辅助机制的复制体，统称为DNA损伤耐受途径，确保 通过动态重塑实现基因组完整性。这些通路的异常可导致染色体 不稳定和包括癌症在内的一系列疾病。绍尔实验室的长期目标是 了解遗传和表观遗传保真度的分子基础，目标是改进治疗 和/或预防疾病。在这项提议中，Schauer实验室将使用一个功能齐全的复制体 由30多个纯多肽重组而成，研究复制体如何绕过经常发生在 基因组，同时加强遗传和表观遗传的完整性代代相传。他们还建议开发 全细胞裂解系统在天然起源的双链DNA上建立活性复制分叉 复制，而不需要在合成分叉上启动复制。DNA损伤耐受机制将 利用生物化学、单分子生物物理学和结构生物学进行研究。它的结构动力学 S阶段的损伤反应将被描述，重点是介导物激酶MRc1和多个 它调节伸长的复制体的方式。Schauer实验室还建议研究时空 跨损伤合成聚合酶挽救病变停滞复制体的机制，以及MRc1和MRc1 而DNA滑动钳的泛素化调节了这种反应。当复制染色质时，核小体 在缺乏组蛋白伴侣蛋白的情况下，对复制分叉进展表现出强烈的阻碍作用。肖尔实验室 将研究重组染色质复制分叉处的组蛋白动力学，重点是调节 组蛋白沉积对称性通过组蛋白伴侣蛋白和各种复制的分子机制- 偶联的组蛋白伴侣本身。将开发工具来跟踪组蛋白的命运和动态 单分子水平。其目标是更好地理解控制表观遗传的过程。 遗传，对于在细胞分裂过程中保持细胞性很重要。最后，Schauer实验室建议研究 复制机制和主动伸长的转录复合体之间的碰撞，因为这些 冲突可能具有高度的诱变性。还将研究rpb4/7异源二聚体的转录调控。 转录将由纯化的蛋白质，或全细胞提取物，或两者的组合重组 二。绍尔实验室正在为这些项目开发生化和单分子工具，这些工具将负担得起 对这些关键过程背后的分子机制的前所未有的一瞥。单分子 荧光共振能量转移(SmFRET)将用于跟踪分子间相互作用， 从而能够表征这些系统的结构动力学。此外，还将开发技术 在单分子水平上跟踪荧光标记蛋白质在双链DNA上的动态运动。 这些研究提供的机械洞察力将有益于医学研究界。

PROJECT SUMMARY Chromosomes are copied by a complex holoenzyme called the replisome. Obstacles are routinely negotiated by the replisome with auxiliary mechanisms, collectively called DNA damage tolerance pathways, that ensure genomic integrity via on-the-fly remodeling. The aberrance of these pathways can lead to chromosome instability and a broad range of diseases including cancer. The Schauer Lab’s long-term goal is to thus understand the molecular basis for genetic and epigenetic fidelity, with the goal of improving the treatment and/or prevention of diseases. In this proposal, the Schauer Lab 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. They also propose to develop whole cell lysate systems to establish active replication forks on double-stranded DNA at natural origins of replication without the need for replication initiation on synthetic forks. DNA damage tolerance mechanisms will be studied using biochemistry, single-molecule biophysics, and structural biology. The structural dynamics of the S-phase damage response will be characterized, with a focus on the mediator kinase Mrc1 and the multiple ways it regulates the elongating replisome. The Schauer Lab also proposes to study the spatiotemporal mechanisms of rescue of lesion-stalled replisomes by translesion synthesis polymerases, and how both Mrc1 and ubiquitination of DNA sliding clamps regulates this response. When replicating chromatin, nucleosomes present a strong block to replication fork progression in the absence of histone chaperones. The Schauer Lab will study histone dynamics at the replication fork in reconstituted chromatin, with a focus on regulation of histone deposition symmetry by histone chaperones and in the molecular mechanisms of various replication- coupled histone chaperones themselves. Tools will be developed to track histone fate and dynamics at the single-molecule level. The goal is to get a better understanding of the processes that control epigenetic inheritance, important for maintaining cellularity during cell division. Finally, the Schauer Lab proposes to study collisions between the replication machinery and an actively elongating transcription complex, since these conflicts can be highly mutagenic. Transcriptional regulation by the rpb4/7 heterodimer will also be studied. Transcription will be reconstituted from either purified proteins, or whole-cell extracts, or a combination of the two. The Schauer Lab is developing biochemical and single-molecule tools for these projects that will afford an unprecedented glimpse into the molecular mechanisms behind these critical processes. Single-molecule fluorescence resonance energy transfer (smFRET) will be employed to track intermolecular interactions, allowing a characterization of the structural dynamics of these systems. Further, technology will be developed to track dynamic motions of fluorescently labeled proteins on double-tethered DNA at the single-molecule level. The mechanistic insight afforded by these studies will be beneficial for the medical research community.