The molecular basis of RECQ4-associated genetic disorders and cancer predispositi

描述（由申请人提供）：预防和修复DNA损伤的功能机制是维持基因组完整性和防止肿瘤发生所必需的。一组属于RecQ家族的蛋白质是与修复DNA损伤剂（如电离辐射和顺铂）引起的病变有关的癌症抑制因子之一。在进化过程中，RecQ基因似乎已经被扩增，并从细菌和酵母中的单个RecQ基因拷贝分化为人类中的五个RecQ同源基因。人类和小鼠模型中不同RECQ蛋白突变引起的临床表型表明，它们在维持基因组完整性和预防癌症方面具有重要且不重叠的作用。由于RECQ解旋酶是高度相互作用的蛋白质，并在体内形成大的蛋白质复合物，因此每个RECQ蛋白的独特功能可能由它们在细胞中形成的特定蛋白质-蛋白质相互作用来定义和调节。为了了解哺乳动物RECQ蛋白如何作为肿瘤抑制因子和我们基因组的守护者，了解每个RECQ蛋白复合物的独特功能至关重要。最近，我们实验室在人类RECQ4解旋酶的研究上取得了一些突破。首先，我们成功地报道了atp依赖性RECQ4解旋酶的体外活性。令人惊讶的是，结构域分析揭示了RECQ4蛋白中两种不同的ATP结合和DNA解绕活动。其次，我们报告了从人类细胞提取物中鉴定出高度纯化的染色质结合RECQ4复合体。我们发现必需的复制体因子MCM10、MCM2-7解旋酶、CDC45和GINS是人类RECQ4的主要相互作用伙伴蛋白。此外，昼夜节律蛋白，TIMELESS和TIPIN对内聚建立和DNA复制进程很重要，是RECQ4复合体的一部分。重要的是，复合体的形成以及RECQ4与复制起源的关联是受细胞周期调控的。我们的研究使我们得出结论，RECQ4是参与人类细胞DNA复制的MCM复制解旋酶复合体的一个组成部分。基于我们的工作，本提案的目标是确定RECQ4复合体在DNA复制，内聚建立和癌症避免中的确切功能。具体目的是：(1)剖析人类RECQ4在DNA复制起始中的作用；(2)建立了RECQ4与TIM-TIPIN异源二聚体在复制叉进展、基因毒性应激反应和内聚建立中的功能关系；(3)癌症相关的RECQ4 （c.1390+2delT）突变分析。

DESCRIPTION (provided by applicant): Functional machineries to prevent and repair DNA damages are required for maintaining genome integrity and preventing tumourigenesis. A set of proteins belonging to the RecQ family is among the cancer suppressors linked to repairing lesions caused by DNA damaging agents, such as ionizing radiation and cisplatin. During the course of evolution, RecQ genes appear to have been amplified and diverged from a single copy of the RecQ gene in bacteria and yeast to five RecQ homologs in humans. The clinical phenotypes caused by mutations in different RECQ proteins in humans and mouse models indicate that they have important and non-overlapping roles in maintaining genomic integrity and cancer prevention. Since RECQ helicases are highly interactive proteins and form large protein complexes in vivo, the unique functions of each of the RECQ proteins are likely defined and regulated by the specific protein-protein interactions they form in cells. To understand how the mammalian RECQ proteins act as tumor suppressors and caretakers for our genome, it is crucial to understand the unique functions of each of the RECQ protein complexes. Recently, our lab has several breakthroughs in the study of human RECQ4 helicase. First, we successfully reported ATP-dependent RECQ4 helicase activity in vitro. Surprisingly, domain analyses uncovered two distinct ATP binding and DNA unwinding activities within the RECQ4 protein. Secondly, we report the identification of a highly purified chromatin bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2-7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Moreover, circadian proteins, TIMELESS and TIPIN important for cohesion establishment and DNA replication progression are part of the RECQ4 complex. Importantly, complex formation and the association of RECQ4 with the replication origin are cell cycle regulated. Our studies allow us to conclude that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells. Based on our work, the goal of this proposal is to define the exact function of RECQ4 complex in DNA replication, cohesion establishment and cancer avoidance. The Specific Aims are: (1) dissecting the role of human RECQ4 in DNA replication initiation; (2) Establishing the functional relationship between RECQ4 and TIM-TIPIN heterodimer in replication fork progression, genotoxic stress response and cohesion establishment; and (3) Analysis of the cancer-associated RECQ4 (c.1390+2delT) mutation. PUBLIC HEALTH RELEVANCE: DNA replication is essential for cell growth, and abnormal DNA synthesis can result in genome instability, mutagenesis and tumorigenesis. Therefore, elucidation of the mechanisms that regulate DNA replication and the molecular functions of the involved proteins, such as RECQ4, are fundamental to our understanding of the cancer development. Crucially, our detailed knowledge on the DNA replication factors will aid in the discovery of new cancer therapy treatments, since inhibiting DNA replication factors effectively prevents cell proliferation.