Title: RuvA and RuvB proteins facilitate the bypass of heterologous DNA insertions during RecA protein-mediated DNA strand exchange.
Abstract: RecA protein-mediated DNA strand exchange between circular single-stranded DNA and linear duplex DNA readily bypasses short (up to 100 base pairs) heterologous inserts in one of the DNA substrates.Larger heterologous inserts are bypassed with decreasing efficiency, and inserts larger than 200 base pairs substantially block RecA-mediated DNA strand exchange.The RuvA and RuvB proteins dramatically facilitate the bypass of larger heterologous inserts.When the RuvA and RuvB proteins are added to an ongoing RecA protein-mediated strand exchange reaction, interior heterologous inserts of 1 kilobase pair are bypassed at significant frequencies.The RuvA, RuvB, and RecA proteins are all required for this activity.Bypass occurs only when homologous sequences are present on both sides of the insert.When the heterologous insert is positioned at either end of the linear duplex substrate, the RuvA and RuvB proteins do not significantly increase product formation in RecA protein-mediated DNA strand exchange reactions.The results suggest an important role for RuvA and RuvB in the bypass of DNA structural barriers during recombinational DNA repair.The RecA protein of Escherichia coli is a central component of the system that promotes homologous genetic recombination and recombinational DNA repair in vivo (Cox, 1991;Roca and Cox, 1990).RecA protein promotes DNA strand exchange reactions in vitro which mimic the principal steps of recombinational DNA repair (Cox and Lehman, 1987;Radding, 1989;Roca and Cox, 1990).The three-strand exchange reaction, shown in Fig. IA, is one of the best characterized in vitro reactions.The first step, which is facilitated by the E. coli SSB protein (Monica1 and Cox, 1990), is the formation of a RecA protein filament on the ssDNA.'The second step is the pairing of a homologous linear duplex DNA with the nucleoprotein filament.Finally, a facilitated unidirectional branch migration, 5' to 3' relative to the ssDNA, leads to formation of nicked circular duplex and displaced single-stranded DNA products.