Huang Y C, Friedman S
Department of Pharmacology, State University of New York Health Science Center, Brooklyn 11203.
J Biol Chem. 1991 Sep 15;266(26):17424-9.
Wild type Escherichia coli cells containing elevated levels of DNA (cytosine-5)methyltransferases have increased sensitivity to the toxic effects of 5-azacytidine. The methyltransferases form tight binding complexes with azacytosine in DNA which could interfere with the recA recBCD repair pathway which is largely responsible for cell survival after treatment with the drug. We therefore determined if these complexes interfered with recA-mediated strand exchange in vitro. 32P-Labeled DNA fragments containing a single EcoRII site, with cytosine in the (-) strand replaced by 5-azacytosine, were prepared. We investigated the effect of the EcoRII methyltransferase on recA-mediated strand exchange with homologous M13 DNA by electrophoresis in agarose gels. In the absence of the methylase the rate and extent of strand exchange of azacytosine-containing DNA is the same as control DNA. In the presence of the methyltransferase strand exchange is inhibited, but some incorporation of duplexes into recA-single-stranded DNA (ssDNA) complexes still occurs. The formation of these complexes is dependent on the length of the fragment 3' to the methylase binding site on the strand complementary to the ssDNA. The greater the length the greater the number of complexes that form. S-Adenosyl-L-methionine, which enhances binding of the methyltransferase to azacytosine-containing DNA, causes an increase in the inhibition of strand exchange and an increase in the number of inactive complexes formed. The complexes can be dissociated with guanidinium chloride which denatures the methyltransferase and leads to release of the (+) strand. The (-) strand remains associated with the ssDNA. This result implies that a plectonemic joint is formed between recA-ssDNA complexes and azacytosine-containing DNA-methyltransferase complexes. However, branch migration in these complexes is inhibited. Denaturation of the methyltransferase allows branch migration to proceed to completion, releasing the (+) strand.
含有高水平DNA（胞嘧啶-5）甲基转移酶的野生型大肠杆菌细胞对5-氮杂胞苷的毒性作用敏感性增加。甲基转移酶与DNA中的氮杂胞嘧啶形成紧密结合复合物，这可能会干扰recA recBCD修复途径，该途径在很大程度上负责药物处理后细胞的存活。因此，我们确定这些复合物是否在体外干扰recA介导的链交换。制备了含有单个EcoRII位点的32P标记DNA片段，其中（-）链中的胞嘧啶被5-氮杂胞嘧啶取代。我们通过琼脂糖凝胶电泳研究了EcoRII甲基转移酶对与同源M13 DNA进行recA介导的链交换的影响。在没有甲基化酶的情况下，含氮杂胞嘧啶DNA的链交换速率和程度与对照DNA相同。在甲基转移酶存在的情况下，链交换受到抑制，但仍有一些双链体掺入recA单链DNA（ssDNA）复合物中。这些复合物的形成取决于与ssDNA互补链上甲基化酶结合位点3'端片段的长度。长度越大，形成的复合物数量越多。S-腺苷-L-甲硫氨酸可增强甲基转移酶与含氮杂胞嘧啶DNA的结合，导致链交换抑制增加和形成的无活性复合物数量增加。这些复合物可以用盐酸胍解离，盐酸胍使甲基转移酶变性并导致（+）链释放。（-）链仍与ssDNA结合。这一结果表明，在recA-ssDNA复合物和含氮杂胞嘧啶DNA-甲基转移酶复合物之间形成了麻花状接头。然而，这些复合物中的分支迁移受到抑制。甲基转移酶的变性允许分支迁移进行到底，释放出（+）链。
