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Studies on the roles of RecQ family proteins in DNA repair and recombination in relation to carcinogenesis and aging

RecQ家族蛋白在与癌变和衰老相关的DNA修复和重组中的作用研究

基本信息

批准号：
09470498
负责人：
ENOMOTO Takemi
金额：
$ 6.27万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470498/
关键词：
RecQ protein DNA helicase Q1 Bloom's syndrome Carcinogenesis Aging DNA topoisomerase III Werner's syndrome Rad18 ワーナー症候群

项目摘要

There exist at least three RecQ homologues in higher eukaryotic cells, DNA helicase Q1, the Bloom's syndrome gene and Werner's syndrome gene product. To clarify the functions of the eukaryotic Rec Q family DNA helicases in DNA repair and recombination and to get an insight into the molecular bases of Bloom's syndrome and Werner's syndrome, we analyzed expression of mRNAs of RecQ family proteins and DNA topoisomerase III which is speculated to function with RecQ helicases, and searched proteins which interact with RecQ family proteins. In addition, we analyzed functional domains of RecQ helicases using gene disruptants of SGS1, which is the yeast RecQ homologue.1. We cloned cDNAs encoding mouse DNA helicase Q1, Bloom's syndrome helicase, DNA topoisomerase IIIalpha and a novel DNA topoisomerase III, IIIbeta (TOP3 beta). We found that messages of these four proteins were highly expressed in the testis. The levels of these mRNAs in the testis increased after birth when the cells in the pachytene phase began to appear and increase.2. We screened for proteins using the yeast two-hybrid system with mouse Wrn as bait and identified three proteins. One is a novel protein, Wip1 (Werner interacting protein 1) and the other two are UBC9 and SUMO-1 (small ubiquitin-related modifier-1), indicating that the function of Werner helicase is regulated by the SUMO- 1 conjugation system.3. We introduced mutations, which were found in the genes of Bloom's syndrome and Werner's syndrome patients, into SGS1 gene and analyzed phenotypes of the yeasts transfected with mutated genes. We found that two different mechanisms are operated in RecQ functions, one requiring DNA helicase activity and one not.

在高等真核细胞中至少存在三个RecQ同源物，分别是DNA解旋酶Q1、Bloom's综合征基因和Werner's综合征基因产物。为了明确真核生物RecQ家族DNA解旋酶在DNA修复和重组中的功能，深入了解Bloom's综合征和Werner's综合征的分子基础，我们分析了RecQ家族蛋白mrna和推测与RecQ解旋酶起作用的DNA拓扑异构酶III的表达，并寻找与RecQ家族蛋白相互作用的蛋白。此外，我们利用酵母RecQ同源物SGS1的基因干扰物分析了RecQ解旋酶的功能域。我们克隆了编码小鼠DNA解旋酶Q1、Bloom's综合征解旋酶、DNA拓扑异构酶IIIalpha和一种新型DNA拓扑异构酶IIIbeta （TOP3 beta）的cdna。我们发现这四种蛋白的信息在睾丸中高度表达。这些mrna的水平在出生后睾丸中开始增加，此时处于粗成期的细胞开始出现并增加。以小鼠Wrn为诱饵，利用酵母双杂交系统进行蛋白筛选，鉴定出3个蛋白。一个是新的蛋白Wip1 (Werner interaction protein 1)，另外两个是UBC9和SUMO-1 (small ubiquinin -related modifier-1)，表明Werner解旋酶的功能受到SUMO-1偶联系统的调控。我们将Bloom's综合征和Werner's综合征患者基因中的突变引入SGS1基因，并分析了转染突变基因的酵母的表型。我们发现在RecQ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。