A role for RNA synthesis in homologous pairing events

Abstract

The relationship between RNA synthesis and homologous pairing in vitro, catalyzed by RecA protein, was examined by using an established strand transfer assay system. When a short DNA duplex is mixed with single-stranded circles, RecA protein promotes the transfer of the minus strand of the duplex onto the complementary region of the plus-strand circle, with the displacement of the plus strand of the duplex. However, if minus-strand RNA is synthesized from the duplex pairing partner, joint molecules containing the RNA transcript, the plus strand of the DNA duplex, and the plus-strand circle are also observed to form. This reaction, which is dependent on RNA polymerase, sequence homology, and RecA protein, produces a joint molecule that can be dissolved by treatment with RNase H but not RNase A. Under these reaction conditions, product molecules form even when the length of shared homology between duplex and circle is reduced to 15 bp.