Binding and repair of O6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context

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Abstract
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 8.4 × 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 × 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 × 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 × 10 5 M −1 ). The k-values (6.5−11.5 × 10 6 M −1 s −1 ) were not significantly dependent on nucleotide sequence. k-values of 5.3 and 4.0 × 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.

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