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预防肿瘤发生所必需的。一组属于RecQ家族的蛋白质是与修复由DNA损伤剂（如电离辐射和顺铂）引起的病变有关的癌症抑制剂之一。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RecQ基因的单拷贝分化为人类中的五个RecQ同源物。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癌症预防方面具有重要且非重叠的作用。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蛋白质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蛋白如何充当我们基因组的肿瘤抑制因子和看护者，了解每个RECQ蛋白复合物的独特功能至关重要。 近年来，本实验室在人RECQ 4解旋酶的研究方面取得了多项突破性进展。首先，我们成功地报道了ATP依赖性RECQ 4解旋酶的体外活性。令人惊讶的是，结构域分析揭示了RECQ 4蛋白内两种不同的ATP结合和DNA解旋活性。其次，我们报告了从人细胞提取物中鉴定出高度纯化的染色质结合RECQ 4复合物。我们发现复制体必需因子MCM 10、MCM 2 -7解旋酶、CDC 45和GINS是RECQ 4的主要相互作用伴侣蛋白。此外，昼夜节律蛋白，TIMELESS和TIPIN对内聚力建立和DNA复制进程很重要，是RECQ 4复合物的一部分。重要的是，复合物的形成和RECQ 4与复制起点的关联是细胞周期调节的。我们的研究使我们能够得出结论，RECQ 4是MCM复制解旋酶复合物参与人类细胞中DNA复制的组成部分。基于我们的工作，本提案的目标是确定RECQ 4复合物在DNA复制、内聚建立和癌症避免中的确切功能。具体目标是：（1）剖析人RECQ 4在DNA复制起始中的作用;（2）建立RECQ 4和TIM-TIPIN异二聚体在复制叉进展、遗传毒性应激反应和内聚力建立中的功能关系;和（3）分析癌症相关RECQ 4（c.1390+2delT）突变。

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.