Mechanism of the BLM/Sgs1 Helicase Complex

描述（由申请人提供）：基因组稳定性的丧失与许多使患者易患癌症的人类疾病相关。DNA解旋酶的RecQ家族是维持基因组稳定性的关键参与者。虽然这些酶参与同源重组以修复双链DNA断裂和停滞的复制叉，但缺乏对这些酶的DNA修复机制的清晰理解。BLM是一种负责Bloom综合征的RecQ解旋酶，在真核生物物种中广泛保守，其中它结合Top3和Rmi 1亚基以形成称为BTR的复合物。在酵母模型系统中，该复合物被称为STR（Sgs 1-Top3-Rmi 1）。本申请提出使用酵母模型系统来确定RecQ解旋酶的N-末端非解旋酶结构域的酶性质。一个新发现的结构域（SSD）是Sgs 1功能所必需的，并显示DNA链交换（SS）活性。SS活性在人BLM、WRN和RecQ 4中是保守的，并且在所有情况下，SSD与卷曲螺旋多聚化结构域相关。在目的1中，我们将使用模型底物测试SSD与Top3-Rmi 1或解旋酶结构域合作以促进DNA解旋和DNA链通过的假设。在目标2中，我们将通过询问SSD是否是体内STR活性所必需的来研究SSD的生理作用。将测定缺乏SSD的Sgs 1蛋白对基因转换和交换的影响。在目标3中，我们将表征多聚化结构域并确定其是否需要补充BLM-/-细胞中的缺陷。由于SS活性是保守的，但SSD显示很少或没有氨基酸序列相似性，我们将确定酵母和人SSD是否结构相似。在目标4中，我们将扩展我们的研究的非解旋酶结构域的SGS 1的特征的第二个域所需的SGS 1功能。该结构域将通过搜索DNA结合活性和通过测试来自人BLM的可比残基是否在酵母中起作用来进行生物化学表征。这些实验的成功完成将揭示RecQ家族的非解旋酶结构域的功能和BTR/STR在体内DNA修复过程中的潜在底物。 公共卫生相关性：基因组不稳定性是癌细胞最常见的特征之一，并且可能是产生转化表型的促发因素。BLM是RecQ DNA解旋酶家族的成员，其作用是确保基因组响应DNA损伤的稳定性。该项目旨在确定BLM使用酵母作为模型系统来抑制不适当重组的酶机制。该项目将利用该模型系统的众所周知的功能，以确定新鉴定的酶活性的作用以及它如何与BLM蛋白复合物中的其他活性合作。因此，这项研究将为维持基因组稳定性的生物学途径提供新的认识。

DESCRIPTION (provided by applicant): Loss of genome stability is associated with a number of human diseases that predispose patients to cancer. The RecQ family of DNA helicases is a key player in the maintenance of genome stability. Although these enzymes participate in homologous recombination to repair double-stranded DNA breaks and stalled replication forks, a clear understanding of the mechanism of DNA repair by these enzymes is lacking. BLM, a RecQ helicase responsible for Bloom's Syndrome, is widely conserved across eukaryotic species where it binds the Top3 and Rmi1 subunits to form a complex referred to as BTR. In the yeast model system this complex is referred to as STR (Sgs1-Top3-Rmi1). This application proposes to use the yeast model system to determine the enzymatic properties of the N-terminal non-helicase domains of RecQ helicases. A newly identified domain (the SSD) is essential for Sgs1 function and displays DNA strand swapping (SS) activity. SS activity is conserved in human BLM, WRN, and RecQ4 and in all cases the SSDs are associated with coiled-coil multimerization domains. In Aim 1 we will test the hypothesis that the SSD cooperates with Top3-Rmi1 or the helicase domain to promote DNA unwinding and DNA strand passage using model substrates. In Aim 2 we will examine the physiological role of the SSD by asking whether it is required for STR activity in vivo. Sgs1 proteins lacking the SSD will be assayed for effects on gene conversion and crossing- over. In Aim 3 we will characterize the multimerization domain and determine whether it is required to complement defects in BLM-/- cells. Because SS activity is conserved, but the SSDs show little or no amino acid sequence similarity, we will determine whether the yeast and human SSDs are structurally similar. In Aim 4 we will extend our studies of the non-helicase domain of Sgs1 to characterize a second domain required for Sgs1 function. The domain will be characterized biochemically by searching for DNA binding activity and by testing whether comparable residues from human BLM function in yeast. Successful completion of these experiments will reveal the function of non-helicase domains of the RecQ family and the potential substrates of BTR/STR during DNA repair in vivo. PUBLIC HEALTH RELEVANCE: Genomic instability is one of the most common characteristics of cancer cells and may be a precipitating factor in generating the transformed phenotype. BLM is a member of RecQ family of DNA helicases that acts to insure genome stability in response to DNA damage. This project seeks to determine the enzyme mechanism that BLM uses to suppress inappropriate recombination using yeast as a model system. The project will exploit well-known features of this model system to determine the role of a newly-identified enzyme activity and how it cooperates with other activities in the BLM protein complex. Thus, this research will provide new understanding about the biological pathways that maintain genome stability.