Lower fidelity of RecA protein catalysed homologous pairing with a superhelical substrate

Abstract

The heteroduplex joint, a splice containing paired strands from each of two DNA molecules is a key feature of homologous genetic recombination. The formation of such a lap joint ensures that heterologous chromosomes do not recombine; presumably the degree of homology determines the frequency of crossing over between related but non-identical chromosomes. On the other hand, according to the current ideas on recombination, the formation of heteroduplex joints is not a stringent process. Genetic evidence supports the view that the classical phenomena of meiotic gene conversion and aberrant meiotic segregation result in part from the inclusion of mismatched base pairs in heteroduplex joints, and the subsequent correction of some of these mismatched pairs before replication. Thus meiotic gene conversion signals one kind of departure from perfectly faithful pairing. The association of increased mutation frequencies with crossing-over is another kind of departure from fidelity in recombination. Little is known about the effect of imperfect homology either on the initial pairing of DNA molecules, or on the formation and extension of heteroduplex joints. The nucleotide sequences of phages 3ΦX174 and G4 are related, but differ by 33% of bases in the coding regions. Here we show using combinations of 3ΦX174 and G4 DNA, that Escherichia coli RecA protein catalyses the formation of joint molecules with many mismatched base pairs, but only if one of the molecules is superhelical. By contrast, in the absence of RecA protein, superhelicity does not cause ΦX174 and G4 DNA to form D-loops spontaneously at any temperature between 37 and 75°C. These observations focus attention on the role of RecA protein in unwinding DNA, and on superhelicity as a factor that can lessen the fidelity of homologous pairing.