Dudás Andrej, Marková Eva, Vlasáková Danusa, Kolman Ada, Bartosová Zdena, Brozmanová Jela, Chovanec Miroslav
Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava 37, Slovak Republic.
Yeast. 2003 Apr 15;20(5):389-96. doi: 10.1002/yea.971.
The Saccharomyces cerevisiae rad52 mutants are sensitive to many DNA damaging agents, mainly to those that induce DNA double-strand breaks (DSBs). In the yeast, DSBs are repaired primarily by homologous recombination (HR). Since almost all HR events are significantly reduced in the rad52 mutant cells, the Rad52 protein is believed to be a key component of HR in S. cerevisiae. Similarly to the S. cerevisiae Rad52 protein, RecA is the main HR protein in Escherichia coli. To address the question of whether the E. coli RecA protein can rescue HR defective phenotype of the rad52 mutants of S. cerevisiae, the recA gene was introduced into the wild-type and rad52 mutant cells. Cell survival and DSBs induction and repair were studied in the RecA-expressing wild-type and rad52 mutant cells after exposure to ionizing radiation (IR) and methyl methanesulphonate (MMS). Here, we show that expression of the E. coli RecA protein partially complemented sensitivity and fully complemented DSB repair defect of the rad52 mutant cells after exposure to IR and MMS. We suggest that in the absence of Rad52, when all endogenous HR mechanisms are knocked out in S. cerevisiae, the heterologous E. coli RecA protein itself presumably takes over the broken DNA.
酿酒酵母rad52突变体对许多DNA损伤剂敏感,主要是对那些诱导DNA双链断裂(DSB)的损伤剂。在酵母中,DSB主要通过同源重组(HR)进行修复。由于在rad52突变体细胞中几乎所有的HR事件都显著减少,因此Rad52蛋白被认为是酿酒酵母中HR的关键组分。与酿酒酵母Rad52蛋白类似,RecA是大肠杆菌中的主要HR蛋白。为了解决大肠杆菌RecA蛋白是否能挽救酿酒酵母rad52突变体的HR缺陷表型这一问题,将recA基因导入野生型和rad52突变体细胞中。在RecA表达的野生型和rad52突变体细胞暴露于电离辐射(IR)和甲基磺酸甲酯(MMS)后,研究了细胞存活以及DSB的诱导和修复情况。在此,我们表明,在暴露于IR和MMS后,大肠杆菌RecA蛋白的表达部分弥补了rad52突变体细胞的敏感性,并且完全弥补了其DSB修复缺陷。我们认为,在没有Rad52的情况下,当酿酒酵母中所有内源性HR机制都被敲除时,异源的大肠杆菌RecA蛋白本身可能会接管断裂的DNA。