Structural Biology of Multi-Domain Proteins and Multi-Protein Machinery in DNA Replication and Repair

项目总结 DNA的忠实复制和对受损DNA的反应，对细胞繁殖和生存至关重要 通过多蛋白质机器的作用。虽然在阐明 细菌和其他低等生物研究中DNA复制和损伤反应的机制， 由于蛋白质序列和结构不保守，因此缺乏关于人类的信息。我们的长- 学期目标是了解人类DNA复制和损伤反应机制的作用。我们的 策略是使用现代结构的全部补充来阐明这些过程的结构基础 生物学工具，并使用一系列生化和生物学方法解释这些信息。我们有 在SV40模型系统中显示，在原点产生单链DNA(SsDNA)的解旋酶 还能主动装载人的单链DNA结合蛋白复制蛋白A(RPA)，而这 在聚合酶-Primase(Pol-Prim)的启动过程中，作用是必不可少的。我们还描述了 模块化RPA蛋白与该单链DNA模板结合时的复杂和动态结构 已经开始解决RPA如何发布模板以将其移交给Pol-Prim的困惑。一旦 在DNA模板上负载底物酶亚基，合成~10nT的RNA， 转移到POLRNADNA亚基上，延伸到最终的~30nT的-DNA引物上。我们的目标是继续 在复制模板时定义模板上的操作轨迹。我们下一阶段的研究是解决 关于释放RPA和将Pol-Prim加载到模板上的基本谜团，计数 引物长度由引物酶决定，从引物酶到POL，再到POL或POL。我们的DNA损伤 反应研究试图了解所涉及的关键蛋白质的作用机制，辨别 疾病相关突变引起的故障的生化基础，并最终利用这一知识 以帮助评估针对这些蛋白质的化疗的潜力。在这里，RPA同样扮演着关键角色， 在这种情况下，在招募伙伴蛋白时，这些蛋白会拖延和重塑复制分叉，并激活重要的信号转导 蛋白质。其目标是获得对相互作用的结构理解和功能验证 RPA和损伤反应蛋白，以及它们在应对受损DNA时的作用。

PROJECT SUMMARY Faithful replication of DNA and response to damaged DNA, essential to cell propagation and survival, proceeds via the action of multi-protein machines. While considerable progress has been made in elucidating the mechanisms of DNA replication and damage response from studies of bacteria and other lower organisms, information on humans is lacking because the protein sequences and structures are not conserved. Our long- term goals are to understand the action of human DNA replication and damage response machinery. Our strategy is to elucidate the structural basis for these processes using the full complement of modern structural biology tools and interpret this information using a range of biochemical and biological approaches. We have shown in the SV40 model system that the helicase that creates the single-stranded DNA (ssDNA) at the origin of replication also actively loads the human ssDNA binding protein replication protein A (RPA), and that this action is essential for DNA priming by DNA polymerase -primase (pol-prim). We have also characterized the complex and dynamic structural architectural of the modular RPA protein as it engages this ssDNA template and have begun to address the quandary of how RPA releases the template to hand off to pol-prim. Once the primase subunits are loaded on the DNA template, an ~10 nt RNA is synthesized and the primed template is transferred to the pol  subunits for extension to the final ~30 nt of RNA-DNA primer. Our goal is to continue to define the trajectory of actions on the template as it is replicated. The next phase of our research is to solve the fundamental mysteries about the release of RPA and loading of pol-prim onto the template, the counting of primer length by primase, and the hand-offs from primase to pol  and in turn to pol  or pol . Our DNA damage response research seeks to understand the mechanism of action of the key proteins involved, discern the biochemical bases for malfunctions caused by disease-associated mutations, and ultimately use this knowledge to help evaluate the potential of chemotherapies targeted to these proteins. Here again, RPA has a critical role, in this case, in recruiting partner proteins that stall and remodel replication forks and activate important signaling proteins. The goal is to obtain structural understanding and functional validation of the interactions between RPA and damage response proteins, and their role in responding to encounters with damaged DNA.