Structural Biology of the DNA Replication Stress Response

项目总结 DNA复制机制不断受到阻碍复制分叉的障碍的挑战。复制 压力和叉子停滞是基因组不稳定的主要来源，这是许多疾病的基础，包括 癌症。复制修复途径称为复制应激反应，用于稳定和重新启动 叉子坏了。然而，人们对这些途径的分子机制知之甚少，部分原因是 缺乏所涉及的蛋白质的结构信息。我们的长期目标是了解分子 复制应激反应的机制以及这些通路如何相互连接以确保忠诚 完成DNA复制。我们的策略是将酶的结构信息和多个蛋白质结合起来 在复制-修复界面工作的复合体及其生物化学和细胞功能。我们是 目前专注于停滞分叉的三个鲜为人知的活动--(1)分叉反转和模板转换 作为稳定受损分叉和重新启动复制的机制，(2)保护不稳定的基本(AP)站点 链切割或诱变旁路；(3)DNA合成的启动。三磷酸腺苷依赖的分叉反转 DNA易位酶HLTF、SMARCAL1和ZRANB3涉及停滞的叉子重塑为四向连接 以防止分叉崩溃并促进复制重启。我们的工作将解决相关知识的严重差距 这些酶如何在受损的叉子上提供独特的修复活动，它们的叉子反转机制，以及 HLTF的泛素连接酶和DNA重塑活性是如何协调和调节分叉逆转的 细胞。其次，我们正在努力了解SOS反应相关多肽酶(SRAP)蛋白是如何 HMCES与单链DNA中的AP位点形成稳定的DNA-蛋白质交联(DPC)，作为一种保护它们免受 复制过程中容易出错的聚合酶和核酸酶。AP部位是DNA损伤最丰富的形式 因此，我们了解细胞如何处理这些强大的复制障碍是至关重要的。我们最近的结构 为进一步的实验理解这本小说背后的化学生物学奠定了基础 修复途径。第三，dna聚合酶α-Primase(PolPrim)是真核生物复制体的核心组成部分。 通过合成确定的RNA-DNA引物，在每个冈崎片段启动从头合成DNA 长度。尽管这一关键活动在复制分叉上很重要，但其作用机制尚不清楚。 我们正在通过捕获polPrim与相关核酸底物的复合体来解决这一知识缺口。 和中间体在其催化循环的不同阶段，并用电子可视化构象状态 显微镜和生物物理方法。中发生的构象动力学的基本知识 DNA从头合成过程中的POL-Prim将是理解酶的协调的第一步 停滞的叉子上的活动。

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.