Studies of WRN, BLM, RecQ4 and Replication Fork Restart

描述（由申请人提供）：本提案中研究的长期目标是了解真核细胞如何协调参与复制、修复和同源重组的大量蛋白质，以忠实地复制其基因组。这个复杂的过程对于维持基因组稳定性至关重要，基因组稳定性的丧失可能导致癌症或过早衰老。最近的研究表明，一个蛋白质家族，RecQ型解旋酶，可能在促进DNA复制中发挥作用。在人类细胞中，有五个RecQ家族成员，其中三个分别在Werner综合征（WRN），Bloom综合征（BLM）或Rothmond-Thompson综合征（RecQ 4）的子集中缺乏。在这项提案中，将采取生物化学的方法来研究WRN，RecQ 4和BLM在复制中的作用，以及更广泛地说，复制叉重新启动所涉及的因素和机制。所使用的模型系统是源自在非洲爪蟾卵提取物中重构的细胞核的核质提取物（NPE）。该体外系统忠实地再现了真核细胞DNA复制的机制和调控。提出了三个具体目标。第一个具体目标是研究FFA-1（非洲爪蟾WRN）和xRecQ 4（非洲爪蟾RecQ 4）在各种定义的DNA底物复制中的作用。第二个具体的目的是研究xBLM（非洲爪蟾布卢姆综合征蛋白）在复制叉组装中的作用，并表征与xBLM相互作用的拓扑异构酶3a（xTopo 3alpha）在复制中的作用。第三个具体目标是利用实验室开发的生化系统，系统地分析停滞的复制叉重新启动的因素和机制。将使用多种方法，包括免疫耗竭、免疫荧光染色、亲和蛋白纯化、重组蛋白表达和tnd生化分级分离来完成拟定的研究。这些研究的结果预计将大大推进对真核DNA复制叉动力学的理解，以及缺陷的RecQ解旋酶如何导致癌症和过早衰老等人类疾病。

DESCRIPTION (provided by applicant): The long-term objective of the studies in this proposal is to understand how eukaryotic cells coordinate the large number of proteins involved in replication, repair, and homologous recombination to faithfully duplicate their genome. This complicated process is essential to the maintenance of genome stability, loss of which can lead to cancer or premature aging. Recent studies suggest that a family of proteins, the RecQ-type helicases, might play a role in facilitating DNA replication. In human cells, there are five RecQ family members, three of which are deficient, respectively, in Werner syndrome (WRN), Bloom syndrome (BLM), or a subset of Rothmond-Thompson syndrome (RecQ4). In this proposal, a biochemical approach will be taken to study the roles of WRN, RecQ4, and BLM in replication and, more broadly, the factors and mechanism involved in replication fork restart. The model system to be used is the nuclearplasmic extracts (NPE) derived from nuclei reconstituted in Xenopus egg extracts. This in vitro system recapitulates faithfully the mechanics and regulation of eukaryotic cellular DNA replication. Three specific aims are proposed. The first specific aim seeks to study the roles of FFA-1 (Xenopus WRN) and xRecQ4 (Xenopus RecQ4) in the replication of various defined DNA substrates. The second specific aim seeks to study the role of xBLM (the Xenopus Bloom syndrome protein) in replication fork assembly and characterize the role of topoisomerase 3a (xTopo 3alpha), which interacts with xBLM, in replication. The third specific aim seeks to systematically analyze the factors and mechanism involved in the restart of stalled replication forks using a biochemical system developed in the lab. A variety of methods, including immunodepletion, immunofluorescence staining, affinity protein purification, recombinant protein expression, and tnd biochemical fractionation will be used to accomplish the proposed studies. The results from these studies are expected to significantly advance the understanding of eukaryotic DNA replication fork dynamics and how defective RecQ helicases lead to human diseases like cancer and premature aging.