Role of the Bloom syndrome DNA helicase BLM in chromosome maintenance mechanisms

布卢姆综合征 DNA 解旋酶 BLM 在染色体维持机制中的作用

• 批准号: 8964799
• 负责人: Kristina Schmidt
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964799
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Adopted; Affect; African; Aging; Alleles; Amino Acids; BLM gene; Bacteria; Binding; Binding Proteins; Biochemical; Bloom Syndrome; C-terminal; Cancer Etiology; Cells; Chimera organism; Chromosomal Instability; Chromosomal Rearrangement; Chromosomal Stability; Chromosome Breakage; Chromosomes; Cleaved cell; Code; Cruciform DNA; DNA; DNA Binding; DNA Damage; DNA Repair; Defect; Delayed Hypersensitivity; Development; Disease; Double Strand Break Repair; Elements; Employee Strikes; Evaluation; Event; Exons; Experimental Designs; Frequencies; Functional disorder; General Population; Genes; Genetic; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Goals; High-Risk Cancer; Human; Hydrolysis; Hypersensitivity; Incidence; Introns; Lead; Libraries; Life; Light; Link; Maintenance; Malignant Neoplasms; Maps; Metabolic; Missense Mutation; Molecular; Mutagenesis; Mutation; N-terminal; NMR Spectroscopy; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; Persons; Phenotype; Population; Predisposition; Proline; Protein Region; Proteins; Refractory; Reporting; Risk Factors; Role; Sequence Analysis; Single Nucleotide Polymorphism; Sister Chromatid Exchange; Structure; Symptoms; Syndrome; Tail; Testing; Variant; Werner Syndrome; Yeasts; base; cancer risk; design; genome integrity; helicase; homologous recombination; in vivo; insight; loss of function; molecular recognition; mutant; novel; population based; public health relevance; repaired; response

 DESCRIPTION (provided by applicant): Defects in many genes with roles in DNA break repair are associated with a striking predisposition to cancer development. One of the most extreme cancer risks is associated with Bloom syndrome (BS) - a chromosome breakage disorder caused by mutations in the RecQ-like DNA helicase BLM. RecQ-like helicases and their role in regulating recombinational DNA repair are conserved from bacteria to humans. Besides BS, defects in RecQ-related genes cause Werner syndrome and Rothmund-Thompson syndrome, which are characterized by accelerated aging and/or increased cancer risk. In addition to BS-associated mutations, 93 missense mutations in the human BLM gene have been reported, but it is unknown which, if any, affect BLM function. It has also been suggested that single nucleotide polymorphisms (SNPs) in introns of BLM that have been associated with higher cancer risk may be linked to coding SNPs in exons of BLM. Using a yeast Sgs1-BLM chimera, we have identified coding SNPs that impair BLM function. They include hypomorphic mutations that define a new class of BLM alleles, not associated with BS, that may increase genome instability, cancer risk and other BS-associated symptoms. One objective of this proposal therefore is to determine the effect of coding SNPs throughout the BLM gene on chromosome stability, DNA break repair and the DNA-damage response, and identify their biochemical defects. In contrast to the helicase core, the >600-residue long N- terminal tails of BLM and the related yeast helicase Sgs1 are disordered and not conserved at the sequence level. They have therefore been refractory to conventional approaches to elucidate their function. It is our hypothesis that the function of the long tails of Sgs1 and BLM arises from structural elements, embedded in disorder, that serve as molecular recognition elements for binding proteins. To test this hypothesis we have designed an approach that combines computational prediction of disorder and interactivity, structure analysis by nuclear magnetic resonance (NMR) spectroscopy, and proline mutagenesis to identify these structural elements and elucidate their importance for BLM and Sgs1 function. Specifically we will (1) use a population- based mutational approach to identify and characterize novel functional motifs in BLM; the ability of BLM variants to rescue high sister-chromatid exchange, double-strand-break-repair defects and hypersensitivity to DNA-damaging agents will be assessed; (2) identify biochemical defects of functionally impaired BLM variants by assessing ATPase, DNA binding, annealing and unwinding activities, and (3) determine disorder-function relationships in the N-terminal tails of Sgs1 and BLM using a combination of (a) NMR to identify regions that are dynamically constrained and may adopt interaction-prone a-helices, (b) proline mutagenesis to disrupt the structural motifs, and (c) functional analysis of novel separation-of-function alleles of SGS1 and BLM in vivo. New insights into function and connectivity of BLM and Sgs1 will elucidate the mechanisms of hyper- recombination and chromosome instability in Bloom syndrome and, generally, in human cancers.

 描述（由申请人提供）：许多在 DNA 断裂修复中发挥作用的基因缺陷与癌症发展的显着倾向相关，最极端的癌症风险之一与布卢姆综合征（BS）有关，这是一种由突变引起的染色体断裂疾病。 RecQ 样 DNA 解旋酶 BLM 及其在调节重组 DNA 修复中的作用在细菌和人类中都是保守的。 Rothmund-Thompson 综合征的特征是加速衰老和/或增加癌症风险 除了 BS 相关突变外，人类 BLM 基因中还报告了 93 种错义突变，但尚不清楚哪些突变（如果有）会影响 BLM 功能。也有人提出，与较高癌症风险相关的 BLM 内含子中的单核苷酸多态性 (SNP) 可能与使用酵母 Sgs1-BLM 的 BLM 外显子中的编码 SNP 相关。嵌合体，我们已经确定了损害 BLM 功能的编码 SNP，它们包括定义一类与 BS 无关的新 BLM 等位基因的亚形突变，可能会增加基因组不稳定性、癌症风险和其他 BS 相关症状。因此，我们的目标是确定 BLM 基因中编码的 SNP 对染色体稳定性、DNA 断裂修复和 DNA 损伤反应的影响，并鉴定其生化缺陷（与解旋酶核心相比）。 BLM 和相关酵母解旋酶 Sgs1 的超过 600 个残基的长 N 末端尾部是无序的，并且在序列水平上不保守，因此难以用常规方法阐明其功能。 Sgs1 和 BLM 的尾部源自嵌入无序中的结构元件，这些结构元件充当结合蛋白的分子识别元件。为了检验这一假设，我们设计了一种方法，该方法将无序的计算预测和交互性、结构分析相结合。具体来说，我们将 (1) 使用基于群体的突变方法来识别和表征 BLM 中的新功能基序；将评估用于挽救高姐妹染色单体交换、双链断裂修复缺陷和对 DNA 损伤剂过敏的 BLM 变体；(2) 识别功能受损的生化缺陷； BLM 变体通过评估 ATP 酶、DNA 结合、退火和解旋活性，以及​​ (3) 结合使用 (a) NMR 来确定 Sgs1 和 BLM 的 N 末端尾部的无序功能关系，以识别动态受限的区域，并可能采用易于相互作用的 a 螺旋，(b) 脯氨酸诱变以破坏结构基序，以及 (c) 体内 SGS1 和 BLM 的新型功能分离等位基因的功能分析。对 BLM 和 Sgs1 的功能和连接性的深入了解将阐明 Bloom 综合征以及人类癌症中的超重组和染色体不稳定性的机制。