Altshuler K B, Hodes C S, Essigmann J M
Department of Chemistry, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Chem Res Toxicol. 1996 Sep;9(6):980-7. doi: 10.1021/tx960062w.
A shuttle vector was constructed in which a single O4-methylthymine (O4MeThy) or O6-methylguanine (O6MeGua) was positioned within a unique NheI restriction site. These lesions are among the many produced when alkylating agents interact with DNA and are the two most widely believed to account for the mutagenicity that follows the alkylation event. The shuttle vectors were transfected in parallel into Chinese hamster ovary cells that were either proficient (mex+) or deficient (mex-) in an endogenous alkyltransferase protein. The vectors integrated into the genome of the host, and the lesions were replicated along with the host chromosome. A portion of the integrated vector encompassing the originally adducted site was subsequently amplified by the polymerase chain reaction from the host genome to mediate analysis of mutation frequency and type. O4MeThy induced a high mutation frequency in both mex- and mex+ cells (28-50% in mex- and 22-42% in mex+). O6MeGua induced a significant but lower level of mutagenesis in the repair-deficient (mex-) cells (7-8.5%) and was not detectably mutagenic in mex+ cells. Mutations induced by the methylated thymine in both cell types were T-->C transitions; the guanine adduct in mex- cells induced G-->A transitions. These results indicate that the O4MeThy lesion is more highly mutagenic than O6MeGua in the same genetic background, and that the former adduct, unlike the latter, does not appear to be repaired to a significant extent by the alkyltransferase or any other mammalian repair enzyme.
构建了一种穿梭载体，其中单个O4-甲基胸腺嘧啶（O4MeThy）或O6-甲基鸟嘌呤（O6MeGua）位于一个独特的NheI限制性酶切位点内。这些损伤是烷化剂与DNA相互作用时产生的众多损伤中的一部分，并且是人们普遍认为的导致烷化事件后致突变性的两种最主要损伤。将穿梭载体平行转染到内源性烷基转移酶蛋白功能正常（mex+）或缺陷（mex-）的中国仓鼠卵巢细胞中。载体整合到宿主基因组中，损伤随宿主染色体一起复制。随后通过聚合酶链反应从宿主基因组中扩增包含最初加合物位点的一部分整合载体，以分析突变频率和类型。O4MeThy在mex-和mex+细胞中均诱导出高突变频率（mex-细胞中为28%-50%，mex+细胞中为22%-42%）。O6MeGua在修复缺陷（mex-）细胞中诱导出显著但较低水平的诱变作用（7%-8.5%），而在mex+细胞中未检测到诱变作用。两种细胞类型中甲基化胸腺嘧啶诱导的突变均为T→C转换；mex-细胞中的鸟嘌呤加合物诱导G→A转换。这些结果表明，在相同遗传背景下，O4MeThy损伤比O6MeGua具有更高的诱变性，并且与后者不同，前者加合物似乎不会被烷基转移酶或任何其他哺乳动物修复酶大量修复。
