Molecular mechanisms of the adaptive response to alkylating agents

将大肠杆菌细胞暴露于相对低水平的烷基化剂会导致形成与烷基化相关的DNA损伤修复相关的酶。一旦这些酶产生，细胞对各种烷基化剂的杀伤和诱变作用变得更有抵抗力。这个过程被称为“适应性反应”。在这一过程中，已知至少有ada、alkA、alkB和aidB四个基因参与，其中ada基因起核心作用。Ada蛋白是Ada基因的产物，作为其自身基因和属于该基因的其他基因的转录调节剂。Ada蛋白具有两种不同的甲基转移酶活性，一种是将甲基从烷基化DNA的甲基磷酸三酯的两种立体异构体中的一种转移到酶的特定半胱氨酸残基（Cys69）上，另一种是将甲基从DNA的O^6-甲基鸟嘌呤或O^4-甲基hymine残基转移到位于c -末端的半胱氨酸残基（Cys321）上。在大肠杆菌中，alkA基因的表达受Ada蛋白控制，Ada蛋白结合到alkA启动子的特定区域，并进一步增强RNA聚合酶全酶与复合物的结合。为了确定Ada蛋白识别的序列，我们在alkA的启动子区域引入了各种碱基替换，并在体内和体外检测了它们对该基因表达的影响。位于转录起始位点-41和-34位点之间的AAAGCAAA序列的碱基变化大大降低了转录起始频率。在足迹实验中，含有该序列的区域受到Ada蛋白的保护，该序列内碱基的改变导致Ada蛋白与启动子结合失败。Ada蛋白很可能识别alkA启动子中的AAAGCAAA序列，并与含有该序列的区域结合，从而允许RNA聚合酶随时进入启动子。Ada蛋白对alkA启动子和Ada启动子的作用机制存在较大差异，尽管两者的表达均受到Ada蛋白的正调控。少

Exposure of Escherichia coli cells to relatively low levels of alkylating agents leads to the formation of enzymes related to the repair of alkylation-associated DNA damage. Once these enzymes are produced, the cell becomes more resistant to both the killing and mutagenic effects of various alkylating agents. This process is termed the "adaptive response". In this process at least four genes, ada, alkA, alkB, and aidB, are known to be involved, among which the ada gene plays a central role. Ada protein, the product of the ada gene, acts as a transcriptional regulator for its own gene and for others belonging to this regulon. The Ada protein carries two distinct methyltransferase activities, one transfers a methyl group from one of two stereoisomers of methylphosphotriesters of alkylated DNA to a specific cysteine residue (Cys69) of the enzyme and the other transfers a methyl group from an O^6-methylguanine or O^4-methylhymine residue of the DNA to a cysteine residue (Cys321) located cl … More ose to the C-terminus of the protein.Expression of the alkA gene in Escherichia coli is controlled by Ada protein, which binds to a specific region of the alkA promoter and enhances further binding of RNA polymerase holoenzyme to the complex. To determine the sequence recognized by the Ada protein, we introduced various base substitutions into the promoter region of alkA and examined their effects on expression of the gene, both in vivo and in vitro. Base changes within the sequence AAAGCAAA, located between positions -41 and -34 from the transcription initiation site, greatly decreased the frequencies of initiation of transcription. In footprinting experiments, the region containing this sequence was protected by the Ada protein and base change within this sequence let to failure of binding of Ada protein to the promoter. It is likely that the Ada protein recognizes the AAAGCAAA sequence in the alkA promoter and binds to the region containing the sequence, thereby allowing ready access of RNA polymerase to the promoter. There are considerable differences between the mechanisms of action of Ada protein on the promoters of alkA and ada, even though the expression of both genes is positively relulatoed by Ada protein. Less