Observation of an anomalously slow association kinetics in the binding of actinomycin D to d(CATGGCCATG)

Abstract

An unusually slow association process which accounts for the bulk of its dichroic changes at 293 nm is observed for d(CAT-GGCC-ATG) when it reacts with actinomycin D (ACTD). This is in contrast to an order of magnitude faster association rates exhibited by oligomers containing a self-complementary tetranucleotide ACTD binding sequence (-TGCA-, -AGCT-, or -CGCG-). The number of drug molecules bound and the melting temperature increase upon ACTD binding are significantly higher for d(CAT-GGCC-ATG) than for other decamers studied. Temperature-dependent spectral measurements of this oligomer in the presence of ACTD suggest additional drug binding prior to denaturation. This particular decamer sequence may be unique, as other decamers containing central -GGCC- sequence and even those differing only by the terminal bases such as d(TAT-GGCC-ATA) and d(GAT-GGCC-ATC) are only weakly binding and do not exhibit such anomalously slow ACTD association kinetics, whereas the dodecamer d(CCAT-GGCC-ATGG) does. CD evidence indicates that, in contrast to the other -GGCC- containing oligomers, both d(CCAT-GGCC-ATGG) and its parent decamer exhibit nonstandard B conformations. The observed slow association kinetics and its interesting D/P dependence are rationalized in terms of a model in which the ACTD molecules initially end-stack and distort the oligomer duplex to a favorable ACTD-binding conformation so that intercalation at the central G-C sequence can occur via DNA breathing.(ABSTRACT TRUNCATED AT 250 WORDS)