Basu A K, McNulty J M, McGregor W G
Department of Chemistry, University of Connecticut, Storrs 06269, USA.
IARC Sci Publ. 1999(150):325-33.
Site-specific studies in several laboratories established that each of the three etheno adducts, 1,N6-ethenoadenine (epsilon A), 3,N4-ethenocytosine (epsilon C) and N2,3-ethenoguanine (N2,3-epsilon G), is mutagenic. In Escherichia coli, epsilon A is only weakly mutagenic in single-stranded DNA (mutation frequency, 0.1%), and epsilon C is at least 20 times more mutagenic than epsilon A. Prior treatment of host cells with ultraviolet irradiation enhances the mutagenic frequency of epsilon C by 30-60%, even when the E. coli is recA. Likewise, enhanced mutagenicity was observed when the host cells lacked 3'-->5' exonuclease activity of DNA polymerase III. epsilon A induces all three base substitutions, but A-->G predominates. epsilon C induces epsilon C-->T and epsilon C-->A substitutions, but only the latter was enhanced after ultraviolet irradiation of host cells. In contrast to the results in bacteria, both epsilon A and epsilon C are potent mutagenic lesions in simian kidney cells, inducing 70 and 81% base substitutions, respectively. In simian kidney cells, epsilon A exclusively induces epsilon A-->G transitions, whereas epsilon C-->A transversions are the major type of mutation induced by epsilon C. Nuclear magnetic resonance (NMR) spectrometry of the four possible pairs containing epsilon C indicated that both epsilon C:G and epsilon C:T pairs are stabilized by hydrogen bonds. Even though the latter forms the most stable pair containing epsilon C, the etheno adduct is in syn alignment. DNA polymerase appears to continue DNA synthesis with a syn-orientated base only in the absence of proofreading exonuclease activity or when ultraviolet irradiation-inducible proteins are present. For epsilon A, only epsilon A:T and epsilon A:G pairs have been studied by NMR, which showed that the former has no hydrogen bond whereas the latter maintains two hydrogen bonds with the etheno base in syn orientation. Determination of the relationship between a particular conformation of epsilon A and its mutagenic activity must await further studies. In a site-specific study of epsilon A with human cell extracts, an 11-mer oligonuclotide with a single epsilon A was inserted into an M13 bacteriophage containing an SV40 origin of replication. This vector was replicated in vitro with human fibroblast cell extracts, and the replicated products were analysed. In this experiment, epsilon A induced predominantly epsilon A-->G transitions but at a mutation frequency of 0.14%.
几个实验室的位点特异性研究表明，三种乙烯基加合物，即1,N6-乙烯基腺嘌呤（εA）、3,N4-乙烯基胞嘧啶（εC）和N2,3-乙烯基鸟嘌呤（N2,3-εG），每一种都具有致突变性。在大肠杆菌中，εA在单链DNA中致突变性较弱（突变频率为0.1%），而εC的致突变性至少是εA的20倍。即使大肠杆菌是recA型的，用紫外线照射宿主细胞也会使εC的致突变频率提高30 - 60%。同样，当宿主细胞缺乏DNA聚合酶III的3'→5'外切核酸酶活性时，也观察到致突变性增强。εA诱导所有三种碱基替换，但A→G占主导。εC诱导εC→T和εC→A替换，但只有后者在宿主细胞紫外线照射后增强。与细菌中的结果相反，εA和εC在猴肾细胞中都是强效的致突变损伤，分别诱导70%和81%的碱基替换。在猴肾细胞中，εA仅诱导εA→G转换，而εC→A颠换是εC诱导的主要突变类型。对包含εC的四种可能碱基对进行核磁共振（NMR）光谱分析表明，εC:G和εC:T碱基对都通过氢键稳定。尽管后者形成了包含εC的最稳定碱基对，但乙烯基加合物处于顺式构象。DNA聚合酶似乎仅在缺乏校对外切核酸酶活性或存在紫外线照射诱导蛋白时，才会以顺式取向的碱基继续DNA合成。对于εA，仅通过NMR研究了εA:T和εA:G碱基对，结果表明前者没有氢键，而后者与乙烯基碱基以顺式取向保持两个氢键。确定εA的特定构象与其致突变活性之间的关系尚需进一步研究。在用人类细胞提取物对εA进行的位点特异性研究中，将含有单个εA的11聚体寡核苷酸插入到含有SV40复制起点的M13噬菌体中。该载体在体外用人成纤维细胞提取物进行复制，并对复制产物进行分析。在这个实验中，εA主要诱导εA→G转换，但突变频率为0.14%。
