Homologous recognition and triplex formation promoted by RecA protein between duplex oligonucleotides and single-stranded DNA

Abstract

RecA protein formed a stable triplex from a 33 bp duplex oligonucleotide and a circular plus strand of M13 DNA when a hairpin connection at the proximal end of the homologous duplex oligonucleotide blocked displacement of the 5' end of its own plus strand. An oligonucleotide with a hairpin connection at the other end yielded five times fewer joints that survived deproteinization, and an ordinary duplex oligonucleotide yielded none. The stability of the three-stranded structure was not attributable to exonucleolytic nibbling of the 3' end of the hairpin oligonucleotide, which could generate a region of stable duplex DNA. In the triplexes, the hairpin duplex became more accessible to copper phenanthroline, exhibited novel sites of cleavage by DNase I, and resisted digestion by Escherichia coli exonuclease I. The enzymatic methylation of only two residues at N-6 adenine and two at N-4 cytosine in the hairpin duplex prior to the pairing reaction lowered the tm of triplexes by 8 deg.C, whereas extensive methylation at N-7 guanine by dimethyl sulfate had no effect. These results are discussed in relation to possible models of triplex DNA.