Binding and repair of O-6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O-6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context
Binding and repair of O-6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O-6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context
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DOI：
10.1093/nar/24.11.2087
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发表时间：
1996-06-01
影响因子：
14.9
通讯作者：
Rajewsky, MF
中科院分区：
文献类型：
--
作者：
Bender, K;Federwisch, M;Rajewsky, MF
双链（ds）寡脱氧核苷酸（29聚体），含有O-6-乙基鸟嘌呤（O-6-EtGua），5'和3'侧接不同的碱基（5 '... TGT..三英尺五英尺公司简介三尺五尺GGT..三英尺五英尺GGG..三英尺五英尺GGA.. 3 '）的体外结合和修复特性。用凝胶阻滞法和单克隆抗体结合实验分别测定了AT与寡聚体的表观结合常数（K-A（app））和O-6-EtGua的修复速率常数（k），当使用含或不含O-6-EtGua的ds-或单链（ss）寡聚物时，在两种底物中均未观察到K-A（app）值的重大差异：天然AT的K-A（app）值分别为7.1和未修饰的，未修饰的和含[O-6-EtGua]的顺式寡聚物的K-A（app）值分别为8.4 × 10（5）M（-1），β-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结合的影响有限; N端截短的AT蛋白质（1.1 × 10（5）M（-1））和天然AT的K-A（app）值具有相同的数量级。此外，K-A（app）几乎不受Cys（145）-甲基化AT（2.0 × 10（5）M（-1））的影响。的k值（6.5-11.5 x 10（6）M（-1）s（-1））不显著依赖于核苷酸序列，使用N-末端截短AT蛋白和修复复制后错配对[O-6-EtGua]获得的k值分别为5.3和4.0 x 10（6）M（-1）s（-1）：低K-A（app），乙基化对K-A（app）鸟嘌呤-O-6的影响可忽略，以及通过改变O-6-EtGua侧翼碱基对K-A（app）和k的轻微调节，所有这些都表明AT与DNA的结合是非特异性的并且主要由离子相互作用介导[在增加的离子强度下降低的K-A（app）和k值]，过量的DNA通过与AT分子的竞争性结合降低了O-6-EtGua在ds-寡聚体中的修复速率，AT与DNA在体内的反应机制有待进一步研究。
Double-stranded (ds) oligodeoxynucleotides (29mers) containing an O-6-ethylguanine (O-6-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 O-6-alkylguanine-DNA alkyltransferase (AT) in vitro, The apparent association constant (K-A(app) Of AT to the oligomers and the repair rate constant for O-6-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 O-6-EtGua were used, no major differences in K-A(app) values were observed with either substrate: K-A(app) values for native AT were 7.1 and unmodified and 8.4 x 10(5) M(-1) respectively, for unmodified and [O-6-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 K-A(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, K-A(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 [O-6-EtGua]: T by native AT, The low K-A(app), the negligible influence on K-A(app) guanine-O-6 of ethylation, and the minor modulation of K-A(app) and k by varying the bases flanking O-6-EtGua, all indicate that the binding of AT to DNA is non-specific and mediated mainly by ionic interactions [reduced K-A(app) and k-values at increased ionic strength], Surplus DNA reduces the rate of O-6-EtGua repair in ds-oligomers by competitive binding of AT molecules, The reaction mechanism of AT with DNA in vivo requires further investigation.