Integrative Structural Biology in DNA Replication and Damage Response

项目摘要 DNA的忠实复制和对异常DNA的反应对细胞繁殖至关重要 和生存我们的长期目标是了解多蛋白质DNA复制和损伤的作用 真核生物复制叉的反应机制。我们的策略是阐明 使用综合结构生物学方法，结合生物化学/生物物理表征， 协作以确定功能含义。该提案的重点是关于以下方面的关键未解决问题： 在复制过程中子链合成的启动，以及复制叉的停滞和重塑， 遇到异常DNA在DNA复制中，进行性聚合酶δ和ε需要短引物 DNA聚合酶α-引物酶（pol-primase）产生的DNA链在模板上起作用。虽然3D 已经确定了聚合物的所有组分和完整的异源四聚体的结构， 只提供了有限的机制的见解，因为全长蛋白质的结构与相关底物 缺乏必要的辅助因子。为了解决这一关键的知识差距，我们建议确定 相关的结构使用Cryo-EM。我们还提议继续努力确定该结构的特征， 4Fe-4S团簇的生物化学性质及其在聚合物中的功能作用。我们将测试和完善我们的 关于以下作用的假说：（i）引发酶4Fe-4S簇氧化还原在调节DNA结合活性中的作用;（ii）引发酶4Fe-4S簇氧化还原在调节DNA结合活性中的作用;（iii）引发酶4Fe-4S簇氧化还原在调节DNA结合活性中的作用;（iv）引发酶4Fe-4S簇氧化还原在调节DNA结合活性中的作用。 polα中的簇在驱动从引物酶的RNA合成到pol α的DNA合成的转变中的作用。 总之，这些研究将解决关于pol-bits如何计算长度的基本问题。 每个步骤的引物以及底物如何从引物酶传递到pol α，然后从pol α传递到pols δ，或 ε。我们的第二个项目解决了关于复制分叉遇到的知识方面的两个关键差距， 异常DNA RPA和Rad 51是两种高度丰富的ssDNA结合蛋白，其在细胞凋亡中具有关键作用。 失速叉的失速、反转和稳定。RPA包被的ssDNA是损伤的起始信号 反应途径，并发挥几个额外的作用，包括招募和指导分叉逆转 ATP马达蛋白SMARCAL 1的活性。我们建议阐明驱动这一机制， 通过在模型车叉上确定RPA和SMARCAL 1的结构，是车叉改造的一个重要方面 使用Cryo-EM的底物复合物。Rad 51在稳定停滞的复制中起重要作用 叉子合作研究导致了RADX的发现和表征，这是一种新的DNA损伤反应 参与调节停滞分叉处Rad 51活性的蛋白质。RADX还与RPA进行物理交互， 这表明RPA-RADX-Rad 51网络在停滞的分叉处运行。我们建议结合结构， RADX的生物物理和功能分析及其与DNA，Rad 51和RPA的相互作用，以澄清 暂停的复制分叉上的RADX角色。我们的两个项目将大大提高对 DNA是如何在真核生物复制叉中加工的，基因组是如何维持和繁殖的。

PROJECT SUMMARY Faithful replication of DNA and response to encounters with aberrant DNA are essential to cell propagation and survival. Our long-term goal is to understand the action of multi-protein DNA replication and damage response machinery at eukaryotic replication forks. Our strategy is to elucidate the structural mechanisms using an integrative structural biology approach, coupled to biochemical/biophysical characterization and collaborations to define functional implications. This proposal focuses on critical unsolved questions about the initiation of daughter strand synthesis in replication, and the stalling and remodeling of replication forks upon encountering aberrant DNA. In DNA replication, the processive polymerases δ and ε require a short primer strand on the template to function, which is generated by DNA polymerase α-primase (pol-prim). Although 3D structures have been determined for all components of pol-prim and the intact heterotetramer, these have provided only limited mechanistic insights because structures of full-length protein with relevant substrates and essential co-factors are lacking. To address this critical gap in knowledge, we propose to determine the relevant structures using Cryo-EM. We also propose to continue working on characterizing the structure, biochemical properties and functional roles of 4Fe-4S clusters in pol-prim. We will test and refine our hypotheses about the role of: (i) primase 4Fe-4S cluster redox in modulating DNA binding activity; (ii) the role of the cluster in polα in driving the transition from RNA synthesis by primase to DNA synthesis by pol α. Together, these studies will solve the fundamental questions about how pol-prim counts the length of the primer at each step and how substrate hand-offs occur from primase to pol α and then from pol α to pols δ or ε. Our second project addresses two critical gaps in knowledge about replication fork encounters with aberrant DNA. RPA and Rad51 are two highly abundant ssDNA binding proteins that have critical roles in the stalling, reversal and stabilization of stalled forks. RPA-coated ssDNA is the initiating signal for damage response pathways and plays several additional roles, including recruiting and directing the fork reversal activity of the ATP motor protein SMARCAL1. We propose to elucidate the mechanisms that drive this important aspect of fork remodeling by determining the structure of the RPA and SMARCAL1 on a model fork substrate complex using Cryo-EM. Rad51 plays an essential role in the stabilization of stalled replication forks. Collaborative studies led to the discovery and characterization of RADX, a new DNA damage response protein involved in regulating the activity of Rad51 at stalled forks. RADX also interacts physically with RPA, suggesting there is a RPA-RADX-Rad51 network operating at stalled forks. We propose combined structural, biophysical and functional analyses of RADX and its interactions with DNA, Rad51 and RPA to clarify the roles of RADX at stalled replication forks. Together, our two projects will greatly enhance understanding of how DNA is processed at eukaryotic replication forks and genomes are maintained and propagated.