Alanazi Mohammed, Leadon Steven A, Mellon Isabel
Department of Biochemistry and Molecular Biology, University of Kentucky, Lexington, KY 40536, USA.
Nucleic Acids Res. 2002 Nov 1;30(21):4583-91. doi: 10.1093/nar/gkf588.
Using a monoclonal antibody that specifically recognizes thymine glycol (Tg) in DNA, we measured the kinetics of the removal of Tg from the genomes of wild-type and repair gene mutant strains of Escherichia coli treated with hydrogen peroxide. Tg is rapidly and efficiently removed from the total genomes of repair-proficient cells in vivo and the removal of Tg is completely dependent on the nth gene that encodes the endonuclease III glycosylase. Hence, it appears that little redundancy in the repair of Tg occurs in vivo, at least under the conditions used here. Moreover, previous studies have found that nth mutants are not sensitive to killing by hydrogen peroxide but xth mutant strains (deficient in the major AP endonuclease, exonuclease III) are sensitive. We find that cell death correlates with the persistence of single-strand breaks rather than the persistence of Tg. We attempted to measure transcription-coupled removal of Tg in the lactose operon using the Tg-specific monoclonal antibody in an immunoprecipitation approach but were not successful in achieving reproducible results. Furthermore, the analysis of transcription-coupled repair in the lactose operon is complicated by potent inhibition of beta-galactosidase expression by hydrogen peroxide.
我们使用一种能特异性识别DNA中胸腺嘧啶乙二醇（Tg）的单克隆抗体，测定了用过氧化氢处理的野生型和修复基因突变型大肠杆菌菌株基因组中Tg的去除动力学。在体内，Tg能从修复功能正常的细胞的总基因组中快速且高效地被去除，并且Tg的去除完全依赖于编码内切核酸酶III糖基化酶的nth基因。因此，至少在此处所用的条件下，体内Tg修复过程中似乎几乎没有冗余现象。此外，先前的研究发现，nth突变体对过氧化氢杀伤不敏感，但xth突变菌株（缺乏主要的AP内切核酸酶，即核酸外切酶III）敏感。我们发现细胞死亡与单链断裂的持续存在相关，而非与Tg的持续存在相关。我们尝试使用Tg特异性单克隆抗体，通过免疫沉淀法测定乳糖操纵子中转录偶联的Tg去除情况，但未成功获得可重复的结果。此外，过氧化氢对β-半乳糖苷酶表达的强烈抑制作用使乳糖操纵子中转录偶联修复的分析变得复杂。
