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

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.