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. 1996 Jun 1;24(11):2087–2094. doi: 10.1093/nar/24.11.2087

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.

K Bender 1, M Federwisch 1, U Loggen 1, P Nehls 1, M F Rajewsky 1
PMCID: PMC145916  PMID: 8668540

Abstract

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.

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Selected References

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