Bypass Mechanisms in Eukaryotic Replication

真核复制中的旁路机制

• 批准号: 10798784
• 负责人: Grant Schauer
• 金额: $ 3.24万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798784
• 关键词: Biochemical; Biochemistry; Biophysics; Bypass; Cell Extracts; Cell division; Cell physiology; Cells; Cellularity; Chromatin; Chromosomal Instability; Chromosomes; Closure by clamp; Communities; Complex; Coupled; DNA; DNA Damage; DNA biosynthesis; DNA replication fork; Deposition; Disease; Ensure; Epigenetic Process; Failure; Generations; Genetic; Genetic Transcription; Genome; Genomics; Goals; Histones; Holoenzymes; Human Pathology; Hybrids; Knowledge; Label; Lesion; Malignant Neoplasms; Mediation; Mediator; Medical Research; Molecular; Molecular Chaperones; Molecular Machines; Motion; Nucleosomes; Pathway interactions; Phosphotransferases; Polymerase; Process; Proteins; RNA; Regulation; Replication Initiation; Replication Origin; Research; S phase; Slide; System; Technology; Transcription Elongation; Transcriptional Regulation; Ubiquitination; design; disorder prevention; ds-DNA; dynamic system; genome integrity; improved; insight; intermolecular interaction; polypeptide; preservation; prevent; protein purification; reconstitution; response; single molecule; single-molecule FRET; spatiotemporal; structural biology; tool

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.

项目概要 染色体由称为复制体的复杂全酶复制。障碍是经常协商的 通过具有辅助机制的复制体，统称为 DNA 损伤耐受途径，确保 通过即时重构实现基因组完整性。这些途径的异常可导致染色体 不稳定和包括癌症在内的多种疾病。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，目标是改善治疗 和/或预防疾病。在该提案中，Schauer 实验室将使用功能齐全的复制体 由 30 多种纯多肽重组而成，用于研究复制体如何绕过经常出现的障碍 基因组，同时加强代际遗传和表观遗传的完整性。他们还提议开发 全细胞裂解物系统在自然起源的双链 DNA 上建立活跃的复制叉 无需在合成叉上启动复制即可进行复制。 DNA损伤耐受机制将 使用生物化学、单分子生物物理学和结构生物学进行研究。结构动力学 S 期损伤反应将被表征，重点是介导激酶 Mrc1 和多重 它调节延伸复制体的方式。绍尔实验室还提议研究时空 通过跨损伤合成聚合酶拯救损伤停滞的复制体的机制，以及 Mrc1 如何 DNA 滑动钳的泛素化调节这种反应。复制染色质时，核小体 在没有组蛋白伴侣的情况下，对复制叉的进展产生强烈的阻碍。绍尔实验室 将研究重组染色质复制叉处的组蛋白动力学，重点关注 组蛋白伴侣的组蛋白沉积对称性以及各种复制的分子机制 耦合组蛋白伴侣本身。将开发工具来追踪组蛋白命运和动态 单分子水平。目标是更好地了解控制表观遗传的过程 遗传，对于细胞分裂过程中维持细胞结构很重要。最后，绍尔实验室建议研究 复制机器和主动延伸的转录复合物之间的碰撞，因为这些 冲突可能具有高度诱变性。还将研究 rpb4/7 异二聚体的转录调控。 转录将由纯化蛋白质、全细胞提取物或两者的组合重建 二。绍尔实验室正在为这些项目开发生化和单分子工具，这些工具将提供 对这些关键过程背后的分子机制进行前所未有的了解。单分子 荧光共振能量转移（smFRET）将用于追踪分子间相互作用， 允许表征这些系统的结构动力学。进一步，技术将得到发展 在单分子水平上追踪双链 DNA 上荧光标记蛋白质的动态运动。 这些研究提供的机制见解将有益于医学研究界。