3,N(4)-ethenocytosine, a highly mutagenic adduct, is a primary substrate for Escherichia coli double-stranded uracil-DNA glycosylase and human mismatch-specific thymine-DNA glycosylase

Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3,N(4)-ethenocytosine (ɛC), 1,N(6)-ethenoadenine (ɛA), N(2),3-ethenoguanine, and 1,N(2)-ethenoguanine cause mutations. The ɛA residues are excised by the human and the Escherichia coli 3-methyladenine-DNA glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repairing ɛC residues have not yet been described. We have identified two homologous proteins present in human cells and E. coli that remove ɛC residues by a DNA glycosylase activity. The human enzyme is an activity of the mismatch-specific thymine-DNA glycosylase (hTDG). The bacterial enzyme is the double-stranded uracil-DNA glycosylase (dsUDG) that is the homologue of the hTDG. In addition to uracil and ɛC-DNA glycosylase activity, the dsUDG protein repairs thymine in a G/T mismatch. The fact that ɛC is recognized and efficiently excised by the E. coli dsUDG and hTDG proteins in vitro suggests that these enzymes may be responsible for the repair of this mutagenic lesion in vivo and be important contributors to genetic stability.

环外DNA加合物（exocyclic DNA adducts）可由多种工业污染物（如致癌物氯乙烯）以及脂质过氧化产生的内源性产物在细胞DNA中生成。嘌呤与嘧啶碱基的乙烯基衍生物，包括3,N(4)-乙烯基胞嘧啶（ɛC）、1,N(6)-乙烯基腺嘌呤（ɛA）、N(2),3-乙烯基鸟嘌呤以及1,N(2)-乙烯基鸟嘌呤，均可诱发突变。ɛA残基可被人类和大肠杆菌的3-甲基腺嘌呤DNA糖苷酶（分别为ANPG蛋白与AlkA蛋白）切除，但目前尚未发现负责修复ɛC残基的酶。本研究鉴定出两种分别存在于人类细胞与大肠杆菌中的同源蛋白，它们可通过DNA糖苷酶活性切除ɛC残基。人类来源的该酶属于错配特异性胸腺嘧啶DNA糖苷酶（hTDG）的活性组分。细菌来源的酶则为双链尿嘧啶DNA糖苷酶（dsUDG），它与hTDG互为同源蛋白。除了具备尿嘧啶与ɛC-DNA糖苷酶活性外，dsUDG蛋白还可修复G/T错配中的胸腺嘧啶。体外实验证实，大肠杆菌dsUDG与hTDG蛋白能够识别并高效切除ɛC残基，这提示上述酶可能在体内负责修复这种诱变损伤，并对维持基因组稳定性具有重要作用。