Mutagenesis by Nucleoside Analogs and its Mechanism

核苷类似物的诱变及其机制

基本信息

批准号：
08044291
负责人：
HAYATSU Hikoya
金额：
$ 1.92万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至无数据
项目状态：
已结题

项目摘要

We have shown that deoxyribosyl dihydropyrimido [4,5-c] [1,2] oxazin-7-one (dP) is a potent mutagen in Escherichia coli and Salmonella typhimurium. In E.coli, this deoxycytidine analog induces both GC-to-AT and AT-to GC transitions. No induced transversions are observed. It is highly mutagenic in a wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. The mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes the phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP,the nucleoside analogue was incorporated into its DNA.The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite both A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analogue and the resulting triphosphate induces a high rate of mutagensis through replicational errors with the error level quite high.N^4-Aminocytidine is a ptently mutagenic nucleoside analog, causing AT-to-GC and GC-to-AT transitions. This mutagenicity was enhanced by the presence of NiCl_2 in the assay on S.typhimurium and E.coli. We also found that N^4-aminocytidine and N^4-aminodeoxycytidine triphosphate can form a complex with Ni^<++>ion. Spectral changes of N^4-aminocytidene induced by Ni^<++> indicated that twe N^4-aminocytidene molecules can be complexed with one Ni^<++>ion. This complex formation may result in the inhibition of the enzymatic degradation of N^4-aminocytidine and/or in the enhancement of the error in DNA replication.

我们已经表明，脱氧核糖基二氢吡啶多[4,5-C] [1,2]恶毒素-7-ONE（DP）是大肠杆菌和Typhimurium沙门氏菌中有效的诱变剂。在大肠杆菌中，这种脱氧类似物类似物诱导GC至AT和AT-GC转变。未观察到诱导的横向。它在野生型大肠杆菌中是高度诱变的，但是在缺乏胸苷激酶的菌株中，这大大降低了。通过甲基丙氨酸的添加可以有效抑制DP诱导的诱变。来自大肠杆菌的部分纯化的胸苷激酶催化了DP的磷酸化至其5'-单磷酸盐。当大肠杆菌在DP存在下生长时，将核苷类似物掺入其DNA中。DNA中DP的含量取决于添加到培养基中的DP的浓度。还使用大肠杆菌DNA聚合酶I大片段研究了DP（DPTP）5'-三磷酸（DPTP）的掺入特性。结果证实，该三磷酸可以在模板中以相似的效率掺入A和G。这表明DP被代谢为胸苷类似物，所得的三磷酸通过复制误差诱导高诱变率，而误差水平很高。N^4-氨基细胞丁胺是一种非常刺激的诱变型核苷类似物，从而导致AT-to-to-gc至gc至gc至gc至at at thats。通过在S. typhimurium和e.coli上的NICL_2中存在NICL_2，这种诱变性得到了增强。我们还发现，n^4-氨基细胞丁胺和N^4-氨基氧基三磷酸可以与Ni^<++>离子形成复合物。 ni^<++>诱导的N^4-氨基丁基的光谱变化表明，Twe N^4-氨基细胞核分子可以与一个Ni^<++>离子复合。这种复合物的形成可能导致抑制N^4-氨基丁胺和/或DNA复制中误差的酶促降解。