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易患癌症的人类疾病有关。 DNA 解旋酶 RecQ 家族在维持基因组稳定性方面发挥着关键作用。尽管这些酶参与同源重组以修复双链 DNA 断裂和停滞的复制叉，但人们对这些酶修复 DNA 的机制仍缺乏清晰的了解。 BLM 是一种导致布卢姆综合征的 RecQ 解旋酶，在真核物种中广泛保守，它与 Top3 和 Rmi1 亚基结合形成称为 BTR 的复合物。在酵母模型系统中，该复合物被称为 STR (Sgs1-Top3-Rmi1)。本申请建议使用酵母模型系统来确定 RecQ 解旋酶 N 端非解旋酶结构域的酶学特性。新发现的结构域 (SSD) 对于 Sgs1 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，并且在所有情况下 SSD 都与卷曲螺旋多聚化域相关。在目标 1 中，我们将测试 SSD 与 Top3-Rmi1 或解旋酶结构域配合使用模型底物促进 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 活性所必需的来检查 SSD 的生理作用。将检测缺乏 SSD 的 Sgs1 蛋白对基因转换和交叉的影响。在目标 3 中，我们将表征多聚化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在结构上是否相似。在目标 4 中，我们将扩展对 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 结合活性并测试来自人类 BLM 的类似残基是否在酵母中发挥作用来进行生化表征。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体内 DNA 修复过程中 BTR/STR 的潜在底物。