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Molecular mechanism of the high incidence of cancer of Bloom's syndrome

布卢姆综合征癌症高发的分子机制

基本信息

批准号：
11138207
负责人：
ENOMOTO Takemi
金额：
$ 4.8万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas (A)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至无数据
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11138207/
关键词：
Bloom's syndrome BLM SCE yeast SGS1 SUMO-1 targeted integration

项目摘要

Bloom's syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We analyzed the functions of yeast Sgs 1, which is the yeast homologue for BLM, as well as those of BLM in higher eukaryotic cells and obtained the following results.1. We made mutated SGS1 genes coding a protein having equivalent missense mutations of BS patients and found that none of the mutated genes could suppress the higher sensitivity to MMS and HU and the elevated SCE of sgs 1 disruptants. Analyses of Sgs 1 functions indicated that the two different mechanisms are operated in Sgs 1 functions, one requiring DNA helicase activity and one not. In addition, genetic analyses indicated that Sgs 1 functions in the down stream of Mec 1 and the upstream of Rad 51.2. We generated a monoclonal antibody specifically recognizing BLM protein and using this antibody, found that BLM is localized in speckled structures in the nucleus. We also obtained a piece of evidence indicating that BLM is associated with SUMO-1. Deletion experiments indicated that the amino acid region 238-586 of BLM is required for the localization in speckled structures.3. We generated BLM^<-/-> and BLM^<-/->/RAD54^<-/-> DT40 cells from the chicken B lymphocyte line DT40 and found that SCE and targeted integration frequencies were increased remarkably in BLM^<-/-> cells. The results obtained with BLM^<-/->/RAD54^<-/-> cells indicated that a large portion of the SCE in BLM^<-/-> cells occurs via homologous recombination and more double strand breaks occur during DNA replication in the absence of BLM and some of these breaks are repaired by homologous recombination.

布卢姆综合征（BS）是一种罕见的遗传性疾病，患者的细胞表现出基因组不稳定性和姐妹染色单体交换（SCE）水平升高。我们对BLM的酵母同源物Sgs 1的功能以及BLM在高等真核细胞中的功能进行了分析，得到以下结果.我们突变了编码BS患者具有等同错义突变的蛋白质的SGS 1基因，发现突变的基因都不能抑制对MMS和HU的较高敏感性以及sgs 1破坏剂的SCE升高。对Sgs 1功能的分析表明，Sgs 1功能中有两种不同的机制，一种需要DNA解旋酶活性，另一种不需要。此外，遗传分析表明，Sgs 1功能在Mec 1的下游和Rad 51.2的上游。我们制备了一种特异性识别BLM蛋白的单克隆抗体，并利用该抗体发现BLM定位于细胞核中的斑点状结构中。我们还获得了一个证据表明，BLM是与SUMO-1。缺失实验表明BLM的238-586位氨基酸区域是斑点状结构定位所必需的.我们从鸡B淋巴细胞系DT 40中获得了BLM^<-/->和BLM^<-/->/RAD 54 ^<-/-> DT 40细胞，发现BLM^<-/->细胞的SCE和靶向整合频率显著增加。用BLM^<-/->/RAD 54 ^<-/->细胞获得的结果表明，BLM^<-/->细胞中的大部分SCE是通过同源重组发生的，在没有BLM的情况下，DNA复制过程中发生更多的双链断裂，其中一些断裂通过同源重组修复。