Purmal A A, Rabow L E, Lampman G W, Cunningham R P, Kow Y W
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405, USA.
Mutat Res. 1996 Dec 2;364(3):193-207. doi: 10.1016/s0921-8777(96)00032-8.
Duplex oligonucleotides containing the base lesion analogs, O-methylhydroxylamine- and O-benzylhydroxylamine-modified abasic (AP) sites, were substrates for the DNA N-glycosylases endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. These N-glycosylases are known to have associated AP lyase activities. In contrast, uracil DNA N-glycosylase, a simple N-glycosylase which does not have an associated AP lyase activity, was unable to recognize the modified AP sites. Endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V recognized the base lesion analogs as N-glycosylases generating intermediary AP sites which were subsequently cleaved by the enzyme-associated AP lyase activities. Kinetic measurements showed that O-alkoxyamine-modified AP sites were poorer substrates than the presumed physiological substrates. For endonuclease III, DNA containing O-methylhydroxyl-amine or O-benzylhydroxylamine was recognized at 12 and 9% of the rate of DNA containing thymine glycol, respectively, under subsaturating substrate concentrations (as determined by relative Vmax/K(m)). Similarly, with formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. DNA containing O-methylhydroxylamine or O-benzylhydroxylamine was recognized at 4-9% of the efficiency of DNA containing N7-methyl formamidopyrimidine or pyrimidine cyclobutane dimers, respectively. Based on the known structures of these base lesion analogs and the substrate specificities of the N-glycosylases, a common mechanism of action is proposed for DNA N-glycosylases with an associated AP lyase activity.
含有碱基损伤类似物、O-甲基羟胺和O-苄基羟胺修饰的无碱基（AP）位点的双链寡核苷酸是DNA N-糖苷酶内切核酸酶III、甲酰胺嘧啶DNA N-糖苷酶和T4内切核酸酶V的底物。已知这些N-糖苷酶具有相关的AP裂解酶活性。相比之下，尿嘧啶DNA N-糖苷酶是一种不具有相关AP裂解酶活性的简单N-糖苷酶，无法识别修饰的AP位点。内切核酸酶III、甲酰胺嘧啶DNA N-糖苷酶和T4内切核酸酶V将碱基损伤类似物识别为产生中间AP位点的N-糖苷酶，随后这些位点被酶相关的AP裂解酶活性切割。动力学测量表明，O-烷氧基胺修饰的AP位点是比假定生理底物更差的底物。对于内切核酸酶III，在亚饱和底物浓度下（由相对Vmax/K(m)确定），含有O-甲基羟胺或O-苄基羟胺的DNA分别以含有胸腺嘧啶二醇的DNA速率的12%和9%被识别。同样，对于甲酰胺嘧啶DNA N-糖苷酶和T4内切核酸酶V，含有O-甲基羟胺或O-苄基羟胺的DNA分别以含有N7-甲基甲酰胺嘧啶或嘧啶环丁烷二聚体的DNA效率的4%-9%被识别。基于这些碱基损伤类似物的已知结构和N-糖苷酶的底物特异性，提出了具有相关AP裂解酶活性的DNA N-糖苷酶的共同作用机制。
