Cole G M, Schild D, Lovett S T, Mortimer R K
Mol Cell Biol. 1987 Mar;7(3):1078-84. doi: 10.1128/mcb.7.3.1078-1084.1987.
The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.
酿酒酵母中的RAD52和RAD54基因参与DNA修复和DNA重组。最近发现RAD54可被X射线诱导，而RAD52则不能。为了进一步研究这些基因的调控，我们利用RAD52和RAD54基因上游的5'区域以及大肠杆菌β-半乳糖苷酶基因的3'末端片段构建了基因融合体。含有这些融合体的整合型或自主复制型质粒的酵母转化体组成性地表达β-半乳糖苷酶活性。此外，RAD54基因融合体在单倍体和二倍体细胞中均可被DNA损伤剂X射线、紫外线和甲基磺酸甲酯诱导，但对热休克无反应。RAD52-lacZ基因融合体对X射线、紫外线辐射或甲基磺酸甲酯几乎没有或没有诱导作用。暴露于此类试剂的细胞中RAD54的典型诱导水平为3至12倍，与先前的mRNA分析结果非常一致。当MATa细胞被α因子阻滞在G1期时，DNA损伤后RAD54仍可被诱导，这表明观察到的诱导与细胞周期无关。使用含有与GAL1启动子融合的EcoRI结构基因的酵母载体，我们表明仅双链断裂在体内就足以诱导RAD54。
