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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

基本信息

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
项目类别：
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

项目摘要

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.