Bronstein S M, Skopek T R, Swenberg J A
Department of Pathology, Duke University, Durham, North Carolina 27710.
Cancer Res. 1992 Apr 1;52(7):2008-11.
The formation and persistence of O6-ethylguanine, O4-ethylthymine, and O2-ethylthymine were quantitated in the genomic DNA of human lymphoblasts exposed to 1.0 mM N-ethyl-N-nitrosourea using immunoslot-blot. The three cell lines used included one which lacks O6-alkylguanine-DNA alkyltransferase, one deficient in nucleotide excision repair, and a third which is competent in both of these repair pathways. The activity of O6-alkylguanine-DNA alkyltransferase was further modulated with O6-benzylguanine, a specific inhibitor of this protein. Repair of the O-ethylated thymines was slow and not related to either DNA repair phenotype. O6-Ethylguanine was repaired with a half-life of about 8 h in cells which expressed both O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair functions. Cells expressing O6-alkylguanine-DNA alkyltransferase activity but lacking nucleotide excision repair showed only slow repair of O6-ethylguanine (half-life of O6-ethylguanine, 43 h), while cells lacking the alkyltransferase showed little or no repair of O6-ethylguanine regardless of nucleotide excision repair activity (half-lives of O6-ethylguanine, 53 to greater than 100 h). We conclude that O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair cooperate in the repair of O6-ethylguanine in human cells.
使用免疫斑点印迹法对暴露于1.0 mM N-乙基-N-亚硝基脲的人淋巴母细胞基因组DNA中O6-乙基鸟嘌呤、O4-乙基胸腺嘧啶和O2-乙基胸腺嘧啶的形成和持久性进行了定量分析。所使用的三种细胞系包括一种缺乏O6-烷基鸟嘌呤-DNA烷基转移酶的细胞系、一种核苷酸切除修复缺陷的细胞系以及第三种在这两种修复途径中均有功能的细胞系。用该蛋白的特异性抑制剂O6-苄基鸟嘌呤进一步调节O6-烷基鸟嘌呤-DNA烷基转移酶的活性。O-乙基化胸腺嘧啶的修复缓慢，且与任何一种DNA修复表型均无关。在同时表达O6-烷基鸟嘌呤-DNA烷基转移酶和核苷酸切除修复功能的细胞中，O6-乙基鸟嘌呤以约8小时的半衰期进行修复。表达O6-烷基鸟嘌呤-DNA烷基转移酶活性但缺乏核苷酸切除修复的细胞中，O6-乙基鸟嘌呤仅缓慢修复（O6-乙基鸟嘌呤的半衰期为43小时），而缺乏烷基转移酶的细胞，无论核苷酸切除修复活性如何，O6-乙基鸟嘌呤几乎没有修复或完全没有修复（O6-乙基鸟嘌呤的半衰期为53至大于100小时）。我们得出结论，O6-烷基鸟嘌呤-DNA烷基转移酶和核苷酸切除修复在人类细胞中O6-乙基鸟嘌呤的修复过程中协同作用。
