Structural Biology of the DNA Replication Stress Response

项目摘要 DNA复制机制不断受到阻碍复制叉的挑战。复制 压力和停滞的分叉是基因组不稳定性的主要来源，这是许多疾病的基础，包括 癌复制修复途径被称为复制应激反应， 损坏的叉子然而，这些途径的分子机制知之甚少，部分原因是 缺乏相关蛋白质的结构信息。我们的长期目标是了解 复制应激反应的机制，以及这些途径如何相互联系，以确保忠实 完成DNA复制。我们的策略是将酶和多蛋白质的结构信息耦合起来 复合物在复制修复界面与其生物化学和细胞功能。我们 目前主要关注三个在停滞分叉中理解不多的活动-（1）分叉反转和模板转换 作为一种机制，以稳定受损的叉和重新启动复制，（2）保护不稳定的脱碱基（AP）位点， 链切割或诱变旁路，和（3）引发DNA合成。由ATP依赖的分叉逆转 DNA移位酶HLTF、SMARCAL 1和ZRANB 3参与将停滞的分叉重塑为四向连接 以防止fork崩溃并促进复制重新启动。我们的工作将解决知识相关的关键差距 这些酶如何在受损的分叉处提供独特的修复活性，它们的分叉逆转机制，以及 泛素连接酶和HLTF的DNA重塑活性是如何协调和调节 细胞其次，我们正在努力了解SOS反应相关肽酶（SRAP）蛋白 HMCES与ssDNA中的AP位点形成稳定的DNA-蛋白质交联（DPC），作为保护它们免受 在复制过程中容易出错的聚合酶和核酸酶。AP位点是最丰富的DNA损伤形式 因此，我们必须了解细胞如何处理这些有效的复制障碍。我们最近的结构 的SRAP DPC形成进一步的实验，以了解这一新的化学生物学的基础 修复途径第三，DNA聚合酶α-引物酶（pol-primase，pol-pyramidase）是真核生物复制体的核心组成部分 通过合成定义的RNA-DNA引物，在每个冈崎片段上启动从头DNA合成 长度尽管复制叉的这一关键活动很重要，但其作用机制尚不清楚。 我们正在通过捕获聚合物与相关核酸底物的复合物来填补这一知识空白 和中间体在其催化循环的各个阶段，并通过电子可视化构象状态 显微镜和生物物理方法。构象动力学的基本知识，发生在 在从头DNA合成过程中的pol-peptide将是理解酶的协调的第一步。 在停滞的岔路口的活动。

PROJECT SUMMARY The DNA replication machinery is constantly challenged by impediments that stall the replication fork. Replication stress and stalled forks are a major source of genomic instability, which underlies a number of diseases including cancer. Replication-repair pathways known as the replication stress response serve to stabilize and restart damaged forks. However, the molecular mechanisms of these pathways are poorly understood, in part because of a dearth of structural information for the proteins involved. Our long term goal is to understand the molecular mechanisms of the replication stress response and how the pathways are interconnected to ensure faithful completion of DNA replication. Our strategy is to couple structural information of the enzymes and multi-protein complexes operating at the replication-repair interface with their biochemistry and cellular functions. We are currently focused on three poorly understood activities at stalled forks—(1) fork reversal and template switching as a mechanism to stabilize damaged forks and restart replication, (2) protection of labile abasic (AP) sites from strand cleavage or mutagenic bypass, and (3) priming of DNA synthesis. Fork reversal by the ATP-dependent DNA translocases HLTF, SMARCAL1, and ZRANB3 involves remodeling of stalled fork into four-way junctions to prevent fork collapse and facilitate replication restart. Our work will address critical gaps in knowledge related to how these enzymes provide unique repair activities at damaged forks, their mechanisms of fork reversal, and how the ubiquitin ligase and DNA remodeling activities of HLTF are coordinated and regulate fork reversal in cells. Secondly, we are working to understand how the SOS Response Associated Peptidase (SRAP) protein HMCES forms a stable DNA-protein crosslink (DPC) with AP sites in ssDNA as a means to protect them from error-prone polymerases and nucleases during replication. AP sites are the most abundant form of DNA damage and thus it is critical that we understand how cells deal with these potent replication blocks. Our recent structure of a SRAP DPC forms the basis for further experiments to understand the chemical biology behind this novel repair pathway. Third, DNA polymerase α-primase (pol-prim) is a core component of the eukaryotic replisome that initiates de novo DNA synthesis at every Okazaki fragment by synthesizing RNA-DNA primers of defined length. Despite the importance of this critical activity at the replication fork, its mechanism of action is unknown. We are addressing this gap in knowledge by trapping complexes of pol-prim with relevant nucleic acid substrates and intermediates at various stages of its catalytic cycle and visualizing conformational states by electron microscopy and biophysical approaches. Fundamental knowledge of the conformational dynamics that occur in pol-prim during de novo DNA synthesis will be the first step toward understanding the coordination of enzymatic activities at stalled forks.