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

在高等真核细胞中至少存在三种RecQ同源物，即DNA解旋酶Q1、Bloom综合征基因和Werner综合征基因产物。为了阐明真核细胞中Rec Q家族DNA解旋酶在DNA修复和重组中的作用，并深入了解Bloom综合征和Werner综合征的分子基础，我们分析了RecQ家族蛋白的mRNAs和推测与RecQ解旋酶有关的DNA拓扑异构酶III的表达，并寻找了与RecQ家族蛋白相互作用的蛋白质。此外，我们利用酵母RecQ同源物SGS1的基因干扰物分析了RecQ解旋酶的功能结构域。我们克隆了编码小鼠DNA解旋酶Q1、布卢姆综合征解旋酶、DNA拓扑异构酶IIIα和新的DNA拓扑异构酶IIIβ(top3β)的cDNA。我们发现，这四种蛋白质的信息在睾丸中高度表达。结果：1.当粗线期细胞开始出现并增多时，睾丸中这些mRNAs的表达水平在出生后开始升高。以小鼠WRN为诱饵，利用酵母双杂交系统筛选蛋白质，鉴定出三种蛋白质。一个是新的Werner相互作用蛋白1(Wip1)，另外两个是UBC9和SUMO-1(小泛素相关修饰物-1)，表明Werner解旋酶的功能受SUMO-1结合系统的调控。我们将在Bloom‘s综合征和Werner’s综合征患者的基因中发现的突变引入SGS1基因，并分析了转导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。

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.