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人类疾病有关，这些疾病使患者容易患上癌症。RECQ家族的DNA解旋酶在维持基因组稳定性方面起着关键作用。尽管这些酶参与同源重组来修复双链DNA断裂和停滞的复制分叉，但对这些酶修复DNA的机制缺乏清楚的了解。BLm是一种导致Bloom综合征的RecQ解旋酶，在真核生物中广泛保守，它与top3和Rmi1亚基结合形成一种称为BTR的复合体。在酵母模型系统中，这种复合体被称为STR(SGS1-top3-Rmi1)。本申请建议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)对SGS1的功能是必不可少的，并显示出DNA链交换(SS)活性。SS活性在人的BLM、WRN和RecQ4中是保守的，并且在所有情况下SSD都与螺旋线圈多聚化结构域相关。在目标1中，我们将使用模型底物验证SSD与top3-Rmi1或解旋酶结构域合作促进DNA解旋和DNA链传递的假设。在目标2中，我们将通过询问SSD是否是体内STR活动所必需的来检查SSD的生理作用。缺少SSD的SGS1蛋白将被检测对基因转化和交换的影响。在目标3中，我们将表征多聚体结构域，并确定它是否需要补充BLM-/-细胞中的缺陷。因为SS的活性是保守的，但SSD显示很少或没有氨基酸序列的相似性，我们将确定酵母和人的SSD在结构上是否相似。在目标4中，我们将扩展我们对SGS1非解旋酶结构域的研究，以表征SGS1功能所需的第二个结构域。该结构域将通过寻找DNA结合活性和测试来自人BLM的可比残基是否在酵母中发挥功能来进行生化表征。这些实验的成功完成将揭示RecQ家族的非解旋酶结构域和BTR/STR在体内DNA修复过程中的潜在底物的功能。 公共卫生相关性：基因组不稳定是癌细胞最常见的特征之一，可能是产生转化表型的一个诱发因素。BLM是DNA解旋酶RecQ家族的成员，该家族在DNA损伤时确保基因组的稳定性。这个项目试图以酵母为模型系统来确定BLM用来抑制不适当重组的酶机制。该项目将利用这个模型系统的众所周知的特征来确定新确定的酶活性的作用，以及它如何与BLM蛋白质复合体中的其他活性合作。因此，这项研究将为维持基因组稳定的生物途径提供新的理解。

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.