Mechanism of the BLM/Sgs1 Helicase Complex

BLM/Sgs1 解旋酶复合物的机制

• 批准号: 8602662
• 负责人: STEVEN J. BRILL
• 金额: $ 4.74万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8602662
• 关键词: Alleles; Amino Acid Sequence; Amino Acids; Binding; Biochemical; Biological; Biological Assay; Biological Models; Bloom Syndrome; Bloom syndrome protein; Cells; Characteristics; Coiled-Coil Domain; Collaborations; Complement; Complex; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Enzymes; DNA-Directed DNA Polymerase; Defect; Enzymes; Eukaryota; Eukaryotic Cell; Excision; Family; Gene Conversion; Genetic; Genetic Crossing Over; Genetic Recombination; Genome Stability; Genomic Instability; Goals; Human; Link; Maintenance; Malignant Neoplasms; Modeling; Molecular Genetics; N-terminal; Pathway interactions; Patients; Phenotype; Physiological; Precipitating Factors; Predisposition; Process; Property; Proteins; RECQL4 gene; Rad52 protein; Research; Role; Rothmund-Thomson syndrome; Saccharomycetales; Series; Sgs1 protein; Structure; Testing; Uncertainty; Werner Syndrome; Yeast Model System; Yeasts; cancer cell; enzyme activity; enzyme mechanism; helicase; homologous recombination; human disease; in vivo; member; mutant; protein complex; repaired; research study; response; tool

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.

描述（由申请人提供）：基因组稳定性的丧失与许多使患者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参与者。尽管这些酶参与了同源重组以修复双链DNA断裂和停滞的复制叉，但缺乏对这些酶修复的DNA修复机制的清晰了解。 BLM是负责Bloom综合征的RECQ解旋酶，在真核物种中广泛保守，它结合了TOP3和RMI1亚基，形成了称为BTR的复合物。在酵母模型系统中，该复合物称为STR（SGS1-TOP3-RMI1）。该应用建议使用酵母模型系统来确定RECQ解旋酶的N末端非螺旋酶结构域的酶促性质。新确定的域（SSD）对于SGS1函数至关重要，并显示DNA链交换（SS）活性。 SS活性在人类BLM，WRN和RECQ4中保守，在所有情况下，SSD都与盘绕螺旋的多层化结构域相关。在AIM 1中，我们将检验以下假设：SSD与Top3-RMI1或解旋酶结构域合作，以促进使用模型底物促进DNA解开和DNA链传递。在AIM 2中，我们将通过询问体内StR活性是否需要SSD的生理作用。缺乏SSD的SGS1蛋白将被分析以对基因转化和跨越的影响。在AIM 3中，我们将表征多聚化域，并确定是否需要补充BLM - / - 单元格中的缺陷。由于SS活性是保守的，但是SSD几乎没有或没有氨基酸序列相似性，因此我们将确定酵母和人类SSD在结构上是否相似。在AIM 4中，我们将扩展对SGS1的非螺旋酶结构域的研究，以表征SGS1功能所需的第二个域。该结构域将通过搜索DNA结合活性以及测试酵母中人类BLM功能的可比较残基来以生化为特征。这些实验的成功完成将揭示RECQ家族的非螺旋酶结构域的功能以及在体内DNA修复过程中BTR/STR的潜在底物的功能。