Liu Liping, Hachey David L, Valadez Gerardo, Williams Kevin M, Guengerich F Peter, Loktionova Natalia A, Kanugula Sreenivas, Pegg Anthony E
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
J Biol Chem. 2004 Feb 6;279(6):4250-9. doi: 10.1074/jbc.M311105200. Epub 2003 Nov 25.
It has been proposed that the DNA repair protein O6-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 Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 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-N7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 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 N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s).
有人提出，DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶通过在其半胱氨酸受体位点与1,2-二溴乙烷反应形成高反应性的半芥子气，从而增加了1,2-二溴乙烷的致突变性，然后该半芥子气可与DNA发生反应（刘，L.，佩格，A.E.，威廉姆斯，K.M.，和根特里奇，F.P.（2002年）《生物化学杂志》277，37920 - 37928）。将大肠杆菌表达的人烷基转移酶与1,2-二溴乙烷和单链寡脱氧核糖核苷酸一起温育，导致形成共价转移酶 - 寡核苷酸复合物。确定的反应顺序为鸟嘌呤>胸腺嘧啶>胞嘧啶>腺嘌呤。对反应产物的胰蛋白酶消化物进行质谱分析表明，一些加合物导致脱嘌呤，释放出与N7位鸟嘌呤交联的Gly136 - Arg147肽，反应位点是活性位点Cys145，这是通过色谱保留时间以及合成肽加合物的串联质谱确定的裂解模式确定的。1,2-二溴乙烷产生的烷基转移酶介导的突变主要是鸟嘌呤到腺嘌呤的转换，但在RpoB基因中此类利福平抗性突变的谱中，20%是鸟嘌呤到胸腺嘧啶的颠换。后者可能源于鸟嘌呤 - N7加合物产生的脱嘌呤位点。存在另一种加合物/致突变途径的证据，因为除了鸟嘌呤N7原子处的DNA加合物外，还获得了其他DNA加合物的证据，以及除了脱嘌呤导致的突变外的其他突变的证据。因此，化学和生物学证据支持至少存在两条1,2-二溴乙烷诱导的致突变性的烷基转移酶依赖性途径，一条涉及烷基转移酶 - S - CH2CH2Br对鸟嘌呤N7的烷基化和脱嘌呤，另一条是尚未表征的系统。
