Structural basis for RPA and DNA primase functions

描述（由申请人提供）：DNA的复制是细胞繁殖和存活所必需的复杂多步骤过程，其通过多蛋白质机器的作用进行。了解复制叉的机制具有很大的影响，因为它是基因组繁殖和维持的最关键位点。虽然从细菌和古细菌的研究中在阐明DNA复制机制方面取得了相当大的进展，但由于蛋白质序列和结构不保守，因此缺乏关于人类复制的信息。我们研究的长期目标是了解人类DNA复制机制的作用。我们的研究目前集中在理解被称为DNA启动的步骤的启动。 我们已经显示了人复制蛋白A（RPA）在复制起点处由SV40解旋酶产生的单链DNA（ssDNA）上的主动加载以及RPA在向DNA引发的过渡中的参与。DNA解旋后，通过引物酶在ssDNA模板上合成初始引物。本文提出的研究旨在深入了解RPA和引发酶如何共同作用以启动引物链的合成。目的1利用小角X射线和中子散射（SAXS，SANS）结合核磁共振谱研究RPA在不同DNA结合态下的结构。目的2解决了与RPA的相互作用，促进引物酶的模板上的加载使用的组合的生化映射和结构分析NMR，建模，SAXS和SANS的作用。一旦引物酶加载到DNA模板上，它就合成约10个核苷酸的引物，然后将引发的模板转移到DNA聚合酶a进行引物延伸。引物酶识别模板和计算引物长度的方法仍然是一个谜。目的3提出通过确定引发酶在不同DNA结合状态下的X射线晶体结构来阐明这些过程的结构基础。总之，这些结果将为ssDNA从RPA到DNA引物酶的传递和RNA引物的计数提供结构基础，这是DNA复制中的关键步骤。

DESCRIPTION (provided by applicant): Replication of DNA is a complex multi-step process essential to cell propagation and survival, which proceeds via the action of multi-protein machines. Understanding the machinery at the replication fork has high impact because it is the most critical site for propagation and maintenance of the genome. While considerable progress has been made in elucidating the mechanisms of DNA replication from studies of bacteria and archae, information on replication in humans is lacking because the protein sequences and structures are not conserved. The long-term goal of our research is to understand the action of the DNA replication machinery in humans. Our research currently focuses on understanding the initiation of the step known as DNA priming. We have shown active loading of human replication protein A (RPA) onto single-stranded DNA (ssDNA) created by the SV40 helicase at the origin of replication and involvement of RPA in the transition to DNA priming. After the DNA is unwound, an initial primer is synthesized on the ssDNA template by primase. The studies proposed here are designed to generate insight into how RPA and primase function together to initiate synthesis of the primer strand. Aim 1 investigates the structure of RPA in different DNA-bound states using a combination of small angle X-ray and neutron scattering (SAXS, SANS) and NMR spectroscopy. Aim 2 addresses the role of interactions with RPA in promoting the loading of primase on the template using a combination of biochemical mapping and structural analyses by NMR, modeling, SAXS and SANS. Once primase is loaded on the DNA template, it synthesizes a ~10 nucleotide primer and then transfers the primed template to DNA polymerase a for primer extension. The means by which primase recognizes the template and counts the length of the primer remains a complete mystery. Aim 3 proposes to elucidate the structural basis for these processes by determining x-ray crystal structures of primase in different DNA bound states. Together, these results will inform the structural basis for the hand-off of ssDNA from RPA to DNA primase and counting of the RNA primer, which are critical steps in the replication of DNA.