Bender K, Federwisch M, Loggen U, Nehls P, Rajewsky M F
Institute of Cell Biology (Cancer Research), University of Essen Medical School, Germany.
Nucleic Acids Res. 1996 Jun 1;24(11):2087-94. doi: 10.1093/nar/24.11.2087.
Double-stranded (ds) oligodeoxynucleotides (29mers) containing an O6-ethylguanine (O6-EtGua) flanked 5' and 3' by different bases (5'..TGT..3'; 5'..CGG..3', 5'..GGT..3'; 5'..GGG..3'; 5'..GGA..3') were synthesized to investigate the binding and repair characteristics of recombinant human O6-alkylguanine-DNA alkyltransferase (AT) in vitro. The apparent association constant (KA(app)) of AT to the oligomers and the repair rate constant for O6-EtGua (k) respectively, were determined by gel retardation and a monoclonal antibody-based filter binding assay. When ds- or single-stranded (ss) oligomers with or without O6-EtGua were used, no major differences in KA(app) values were observed with either substrate: KA(app) values for native AT were 7.1 and 8.4 x 10(5) M(-1) respectively, for unmodified and [O6-EtGua]-containing ds-oligomers. The corresponding values for ss-oligomers were 1.0 and 4.9 x 10(5) M(-1). The N-terminal first 56 amino acids of AT only exert a limited influence on DNA binding; the KA(app) values for an N-terminally truncated AT protein (1.1 x 10(5) M(-1)) and native AT were of the same order. Moreover, KA(app) was hardly affected by Cys(145)-methylated AT (2.0 x 10(5) M(-1)). The k-values (6.5-11.5 x 10(6) M(-1)s(-1)) were not significantly dependent on nucleotide sequence. k-values of 5.3 and 4.0 x 10(6) M(-1)s(-1) respectively, were obtained with the N-terminally truncated AT protein and for repair of the postreplicative mispair [O6-EtGua]: T by native AT. The low KA(app), the negligible influence on O6 of ethylation, and the minor modulation KA(app) and k by varying the bases flanking O6-EtGua, all indicate that the binding of AT to DNA is non-specific and mediated mainly by ionic interactions [reduced KA(app) and k-values at increased ionic strength]. Surplus DNA reduces the rate of O6-EtGua repair in ds-oligomers by competitive binding of AT molecules. The reaction mechanism of AT with DNA in vivo requires further investigation.
合成了双链（ds）寡聚脱氧核苷酸（29聚体），其含有的O6-乙基鸟嘌呤（O6-EtGua）在5'和3'端两侧为不同碱基（5'..TGT..3'；5'..CGG..3'，5'..GGT..3'；5'..GGG..3'；5'..GGA..3'），以研究重组人O6-烷基鸟嘌呤-DNA烷基转移酶（AT）在体外的结合和修复特性。通过凝胶阻滞和基于单克隆抗体的滤膜结合试验分别测定了AT与寡聚体的表观缔合常数（KA(app)）和O6-EtGua的修复速率常数（k）。当使用含或不含O6-EtGua的ds-或单链（ss）寡聚体时，两种底物的KA(app)值均未观察到明显差异：天然AT对未修饰和含[O6-EtGua]的ds-寡聚体的KA(app)值分别为7.1和8.4×10(5) M(-1)。ss-寡聚体的相应值为1.0和4.9×10(5) M(-1)。AT的N端前56个氨基酸对DNA结合仅产生有限影响；N端截短的AT蛋白（1.1×10(5) M(-1)）和天然AT的KA(app)值处于同一水平。此外，KA(app)几乎不受半胱氨酸（145）-甲基化AT（2.0×10(5) M(-1)）的影响。k值（6.5 - 11.5×10(6) M(-1)s(-1)）对核苷酸序列没有显著依赖性。N端截短的AT蛋白对复制后错配[O6-EtGua]：T的修复以及天然AT的修复分别获得k值为5.3和4.0×10(6) M(-1)s(-1)。低KA(app)、乙基化对O6的影响可忽略不计以及通过改变O6-EtGua两侧碱基对KA(app)和k的微小调节，均表明AT与DNA的结合是非特异性的，主要由离子相互作用介导[在离子强度增加时KA(app)和k值降低]。过量的DNA通过AT分子的竞争性结合降低了ds-寡聚体中O6-EtGua的修复速率。AT与体内DNA的反应机制需要进一步研究。
