Bypass Mechanisms in Eukaryotic Replication

真核复制中的旁路机制

• 批准号: 10500889
• 负责人: Grant Schauer
• 金额: $ 36.06万
• 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/10500889
• 关键词: Biochemical; Biochemistry; Biophysics; Bypass; Cell Extracts; Cell division; Cell physiology; Cells; Cellularity; Chromatin; Chromosomal Instability; Chromosomes; Closure by clamp; Communities; Complex; Conflict (Psychology); 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; Lead; Lesion; Malignant Neoplasms; Mediation; Mediator of activation protein; 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; Transcriptional Regulation; Ubiquitination; design; disorder prevention; ds-DNA; genome integrity; improved; insight; intermolecular interaction; polypeptide; preservation; prevent; 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.

项目摘要 染色体是由一种叫做复制体的复杂全酶复制的。障碍是例行谈判 复制体与辅助机制，统称为DNA损伤耐受途径，确保 基因组的完整性。这些通路的异常可导致染色体畸变 不稳定和包括癌症在内的广泛疾病。Schauer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，以改善治疗 和/或预防疾病。在这个提议中，Schauer实验室将使用一个功能齐全的复制体 从30多个纯多肽重组，研究复制体如何绕过在细胞中经常发生的障碍， 基因组，同时加强跨代遗传和表观遗传的完整性。他们还建议发展 全细胞裂解物系统，以在双链DNA的天然起点处建立活性复制叉。 不需要在合成叉上启动复制。DNA损伤耐受机制 使用生物化学，单分子生物物理学和结构生物学进行研究。的结构动力学 S期损伤反应将被表征，重点是介体激酶Mrc 1和多个 调节延长复制体的方式。Schauer实验室还建议研究时空 通过跨损伤合成聚合酶拯救损伤停滞复制体的机制，以及Mrc 1 而DNA滑动夹的泛素化调节了这种反应。当复制染色质时，核小体 在没有组蛋白伴侣的情况下，对复制叉的进展有很强的阻断作用。Schauer实验室 将研究重组染色质复制叉的组蛋白动力学，重点是调节 组蛋白伴侣的组蛋白沉积对称性和各种复制的分子机制- 与组蛋白伴侣结合。将开发工具来跟踪组蛋白的命运和动态， 单分子水平。目的是更好地了解控制表观遗传的过程 遗传，在细胞分裂过程中对维持细胞结构很重要。最后，Schauer实验室建议研究 复制机制和主动延伸的转录复合物之间的碰撞，因为这些 冲突可能是高度突变的。还将研究rpb 4/7异二聚体的转录调控。 转录将从纯化的蛋白质或全细胞提取物或其组合重建。 两个. Schauer实验室正在为这些项目开发生物化学和单分子工具， 前所未有地瞥见了这些关键过程背后的分子机制。单分子 将使用荧光共振能量转移（smFRET）来跟踪分子间相互作用， 允许表征这些系统的结构动力学。此外，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利于医学研究界。