Integrative Structural Biology in DNA Replication and Damage Response

项目总结 DNA的忠实复制和对遇到异常DNA的反应是细胞繁殖所必需的 和生存。我们的长期目标是了解多蛋白DNA复制和损伤的作用 真核生物复制分叉的反应机制。我们的策略是阐明结构机制 使用综合结构生物学方法，结合生化/生物物理表征和 定义功能含义的协作。这项提案的重点是关于 复制中子链合成的启动，以及复制叉子的停滞和重塑 遇到异常的DNA。在dna复制中，过程性聚合酶δ和ε需要一个短的引物。 链在模板上发挥作用，这是由α聚合酶产生的DNA-Primase(PolPrim)。虽然是3D 已经确定了polPrim和完整的杂四聚体的所有组分的结构，这些 仅提供了有限的机制见解，因为全长蛋白质与相关底物的结构 而且缺乏必要的辅助因素。为了解决这一关键的知识差距，我们建议确定 使用Cryo-EM的相关结构。我们还建议继续研究该结构的特征， Pol-Prim中4Fe-4S团簇的生化性质和功能作用。我们将测试和完善我们的 关于：(I)Primase 4Fe-4S簇氧化还原在调节DNA结合活性中的作用的假设；(Ii)作用 Polα中的簇在推动从引物合成到α合成的转变中起着重要的作用。 总而言之，这些研究将解决关于POL-PRIM如何计算 以及底物如何从底物转移到POLα，然后从POLα转移到POLSδ或 ε.我们的第二个项目解决了有关复制分叉遇到的两个关键知识缺口 异常的DNA。RPA和RAD51是两种高度丰富的单链DNA结合蛋白，在 失速叉子的失速、倒车和稳定。RPA包被的单链DNA是损伤的起始信号 反应途径，并扮演几个额外的角色，包括招募和指导分叉逆转 ATP马达蛋白SMARCAL1的活性。我们建议阐明驱动这一现象的机制 通过确定模型叉子上的RPA和SMARCAL1的结构来重塑叉子的重要方面 使用Cryo-EM的基质复合体。RAD51在停滞复制的稳定中起着至关重要的作用 叉子。合作研究导致了一种新的DNA损伤反应RADX的发现和表征 参与调节失速叉子上RAD51活性的蛋白质。RADX还与RPA进行物理交互， 这表明有一个RPA-RADX-RAD51网络在停滞的分叉上运行。我们建议组合结构， RADX的生物物理和功能分析及其与DNA、RAD51和RPA的相互作用 RADX在停滞不前的复制分叉中的角色。我们的两个项目将极大地增进对 DNA是如何在真核复制叉处处理的，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.