RECOGNITION OF SPECIFIC DNA-SEQUENCES BY MITOMYCIN-C FOR ALKYLATION
RECOGNITION OF SPECIFIC DNA-SEQUENCES BY MITOMYCIN-C FOR ALKYLATION
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DOI：
10.1021/bi00120a016
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发表时间：
1992-02-11
期刊：
影响因子：
2.9
通讯作者：
TOMASZ, M
中科院分区：
文献类型：
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作者：
KUMAR, S;LIPMAN, R;TOMASZ, M
合成的寡聚脱氧核苷酸与丝裂霉素C(MC)在限制MC单官能烷基化活性的条件下反应。通过将反应混合物酶解成未反应的核苷和烷基化产物MC-脱氧鸟苷加合物(2)，测定了可变序列寡核苷酸的单官能团烷基化产率，然后用高效液相色谱法进行了定量分析。2的相对产率反映了相对单烷基化反应活性。在具有5‘-NGN’序列的一系列寡核苷酸中进行了比较，其中5‘-碱基变化，而3’-碱基保持不变，如T。在Na2S2O4活化条件下，5‘-CG序列的产率显著提高：36%，而5’-AG和5‘-TG的产率分别为2%和4.1%。5‘-GG序列也表现出较高的反应活性，但幅度较小(14.7%)。这些增强是针对寡核苷酸的双链状态的。用NADPH：细胞色素c还原酶作还原剂也得到了类似的结果。被酸性pH激活的MC也表现出5‘-CG烷基化的特异性。Na2S2O4激活的10-脱氨基甲酰-MC表现出与MC相同的5‘-CG特异性。5‘-CG位点上相反链上的脱氧鸟苷被脱氧肌苷取代后，烷基化的增强作用消失。在5‘-GG部位的这种替换也有类似的效果。研究发现，鸟嘌呤的碱基3‘对G在5’-CG序列上的反应活性的增强只有相对温和的调节作用。这种3‘碱基效应似乎独立于5’-NGN‘三联体的5’碱基。反应级数为3‘-(C&gT；T>；G>；A)。主要基于肌苷取代的结果，提出了MC使DNA烷基化的主要5‘-CG(以及次要的5’-GG)专一性的解释：在5‘-CG上，相反链上的2-氨基与被激活的MC的10-0原子之间形成了一个氢键，促进了该序列上的烷基化。类似的机制也适用于5‘-GG位点。MC单烷基化DNA的5‘-CG和5’-GG位点与两个交联点(CG-CG的链间交联点和GG.CC的链内交联点)相吻合。单烷基化的特异性可能是一种分子进化机制，引导MC优先选择位于DNA可交叉连接序列中的鸟嘌呤。
Synthetic oligodeoxyribonucleotides were reacted with mitomycin C (MC) under conditions which restricted MC to monofunctional alkylating activity. The yields of monofunctional alkylation of oligonucleotides with variable sequence were determined by enzymatic digestion of the reaction mixture to unreacted nucleosides and the product of alkylation, a MC-deoxyguanosine adduct (2), followed by quantitative analysis by HPLC. The relative yields of 2 reflected relative monoalkylation reactivities. They were compared in a series of oligonucleotides having the sequence 5'-NGN' in which the 5'-base was varied while the 3'-base was kept constant as T. Under Na2S2O4 activation conditions a striking enhancement of the yield was observed at the 5'-CG sequence: 36%, compared to 2% at 5'-AG and 4.1% at 5'-TG. The 5'-GG sequence also showed enhanced reactivity although to a lesser extent (14.7%). The enhancements were specific to the duplex state of the oligonucleotides. Using NADPH:cytochrome c reductase as the reducing agent gave similar results. MC activated by acidic pH also displayed 5'-CG alkylation specificity. 10-Decarbamoyl-MC activated by Na2S2O4 showed the same 5'-CG specificity as MC. Replacement of deoxyguanosine by deoxyinosine in the opposite strand at a 5'-CG site abolished the enhancement of alkylation. Such replacement at a 5'-GG site had a similar effect. It was found that the base 3; to the guanine had only a relatively modest modulating effect on the enhanced reactivity of the G at the 5'-CG sequence. This 3'-base effect appeared to be independent of the 5'-base of the 5'-NGN' triplet. The order of reactivity is 3'-(C > T > G > A). An explanation is proposed for the dominating 5'-CG (and, to a lesser extent, 5'-GG) specificity of the alkylation of DNA by MC, based primarily on the results of the inosine substitutions: At 5'-CG a H-bond is formed between the 2-amino group of guanine in the opposite strand and the 10-0 atom of activated MC, facilitating alkylation at such sequence. An analogous mechanism applies at the 5'-GG site. The 5'-CG and 5'-GG sites of DNA monoalkylation by MC coincide with the two cross-linkable sites (interstrand cross-link at CG-CG and intrastrand cross-link at GG.CC). The monoalkylation specificity may be a molecular evolutionary device to guide MC preferentially to guanines located in cross-linkable sequences of DNA.