Characterization of a mutagenic DNA adduct formed from 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase
Characterization of a mutagenic DNA adduct formed from 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase
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DOI：
10.1074/jbc.m311105200
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发表时间：
2004-02-06
影响因子：
4.8
通讯作者：
Pegg, AE
中科院分区：
文献类型：
--
作者：
Liu, LP;Hachey, DL;Pegg, AE
DNA修复蛋白O-6-烷基鸟嘌呤-DNA烷基转移酶通过与1，2-二溴乙烷在半胱氨酸受体处反应形成高活性的半芥末，从而增加其致突变性，从而与DNA发生反应(Liu，L.，Pegg，A.E.，Williams，K.M.，and Guengerich，F.P.(2002)J.Biol)。化学。277、37920-37928)。大肠杆菌表达的人烷基转移酶与1，2-二溴乙烷和单链寡核苷酸孵育后形成共价转移寡核苷酸复合体。测定的反应级数为：Gua>；Thy>；Cyt>；Ade。对反应产物的胰酶消化进行的质谱分析表明，一些加合物通过释放N-7位的Gly(136)-Arg(147)交联肽与GUA发生脱氢反应，反应的中心是由层析保留时间确定的活性中心Cys(145)，以及由合成的多肽加合物的串联质谱仪确定的裂解模式。1，2-二溴乙烷引起的烷基转移酶介导的突变主要是Gua到Ade的转变，但在这种RpoB基因的利福平耐药突变中，有20%是Gua到Thy的颠换。后者很可能来自GUA-N-7加合物产生的无嘌呤位置。支持额外的加合物/突变途径，因为获得了DNA加合物和突变的证据，而不是在Gua N-7原子上的加合物，也不是那些可归因于去嘌呤的突变。因此，化学和生物学证据支持1，2-二溴乙烷致突变至少存在两条依赖于烷基转移酶的途径，一条涉及烷基转移酶-S-CH2CH2Br的GUAN-7烷基化和去嘌呤作用，另一条途径尚未确定(S)。
It has been proposed that the DNA repair protein O-6-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly(136)-Arg(147) peptide cross-linked to a Gua at the N-7 position, with the site of reaction being the active site Cys(145) as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N-7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N-7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N-7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s).