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有关。DNA解旋酶的RecQ家族是维持基因组稳定性的关键角色。虽然这些酶参与同源重组修复双链DNA断裂和停滞的复制叉，但对这些酶修复DNA的机制还缺乏明确的认识。BLM是一种导致布鲁姆综合征的RecQ解旋酶，在真核生物物种中广泛保守，它与Top3和rm1亚基结合形成称为BTR的复合物。在酵母模型系统中，这种复合物被称为STR （Sgs1-Top3-Rmi1）。本应用程序建议使用酵母模型系统来确定RecQ解旋酶的n端非解旋酶结构域的酶性质。一个新发现的结构域（SSD）对Sgs1的功能至关重要，并显示DNA链交换（SS）活性。SS活性在人类BLM、WRN和RecQ4中是保守的，在所有情况下，ssd都与线圈多聚域相关。在Aim 1中，我们将使用模型底物测试SSD与Top3-Rmi1或解旋酶结构域合作以促进DNA解绕和DNA链通道的假设。在目标2中，我们将通过询问SSD是否为体内STR活动所必需来检查SSD的生理作用。将检测缺乏SSD的Sgs1蛋白对基因转化和杂交的影响。在Aim 3中，我们将描述多聚结构域，并确定是否需要它来补充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类ssd是否在结构上相似。在Aim 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结合活性和测试人类BLM的类似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家族非解旋酶结构域的功能以及BTR/STR在体内DNA修复过程中的潜在底物。

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.