Multiple non-B-DNA conformations of polypurine.cntdot.polypyrimidine sequences in plasmids

Abstract

A polypurine.polypyrimidine (Pur.Pyr) sequence with a central interruption in a plasmid can adopt multiple non-B-DNA conformations depending on the conditions as revealed by specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) and two-dimensional electrophoresis. The relatively long mirror repeat Pur.Pyr sequences (GAA)9TTC(GAA)8 and (GGA)9TCC(GGA)8 form single canonical intramolecular triplexes at pH 7.0-6.0 in negatively supercoiled plasmids as isolated from Escherichia coli. With a lowering of the pH and/or an increase in the degree of negative supercoiling, these sequences undergo a novel conformational change as revealed by diethyl pyrocarbonate hypermodification of adenines in the middle of the polypurine strand and OsO4 reaction with thymines in the center and the quarter points of the polypyrimidine strand. To evaluate this structure, a family of related Pur.Pyr sequences were cloned and studied. The non mirror repeat sequence (GGA)9TCC(GAA)8 forms a non-B conformation only under acidic pH conditions, but the structural properties are different from those of the mirror repeat sequences. Furthermore, when the central interruptions of a mirror repeat sequence were increased from 3 to 9 bp, two canonical triplexes formed independently at pH 5.0 [at the (GAA)9 and (GAA)8 regions in the sequence (GAA)9TTAATTCGC(GAA)8]. Thus, if an interruption is sufficiently long, the two halves of the Pur.Pyr sequence do not interact with each other. Novel types of folded DNA geometries which explain these results are described.