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

 描述(由申请人提供)：许多在DNA断裂修复中起作用的基因的缺陷与癌症发展的显著易感性有关。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RecQ样DNA解旋酶BLM突变引起的染色体断裂疾病。RECQ类解旋酶及其在调节重组DNA修复中的作用从细菌到人类都是保守的。除了BS，RecQ相关基因的缺陷还会导致Werner综合征和Rothmund-Thompson综合征，这两种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，已经报道了93个人类BLM基因的错义突变，但尚不清楚是否有影响BLM功能的错义突变。也有研究表明，BLM内含子中的单核苷酸多态(SNPs)可能与BLM外显子中的编码SNPs有关，这些内含子与癌症风险相关。利用酵母SGS1-BLM嵌合体，我们已经鉴定出破坏BLM功能的编码SNPs。它们包括亚型突变，这些突变定义了一类与BS无关的新的BLM等位基因，可能会增加基因组的不稳定性、癌症风险和其他BS相关症状。因此，这项建议的一个目标是确定编码整个BLM基因的SNPs对染色体稳定性、DNA断裂修复和DNA损伤反应的影响，并确定它们的生化缺陷。与解旋酶核心相反，BLM的&gt；600个残基的N端长尾巴和相关的酵母解旋酶SGs1在序列水平上是无序的，不是保守的。因此，它们一直难以用传统的方法来阐明它们的功能。我们的假设是，SGS1和BLM的长尾的功能来自嵌入在无序中的结构元件，这些结构元件作为结合蛋白质的分子识别元件。为了验证这一假说，我们设计了一种结合无序和相互作用的计算预测、核磁共振(核磁共振)谱结构分析和脯氨酸突变的方法来识别这些结构元件，并阐明它们对BLM和SGS1功能的重要性。具体地说，我们将(1)使用基于群体的突变方法来鉴定和表征BLM中的新功能基序；将评估BLM变体拯救高姐妹染色单体交换、双链断裂修复缺陷和对DNA损伤剂的超敏反应的能力；(2)通过评估ATPase、DNA结合、退火和解卷活性来确定功能受损的BLM变异体的生化缺陷，以及(3)结合使用(A)核磁共振来确定动态受限并可能采用相互作用倾向的a-螺旋的区域，(B)用Pro突变来破坏结构基序，以及(C)在体内对SGS1和BLM的新的分离功能等位基因进行功能分析。对BLM和SGS1功能和连接的新见解将阐明Bloom综合征和一般人类癌症中超重组和染色体不稳定的机制。

 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.