IN-VITRO RECONSTITUTION OF HOMOLOGOUS RECOMBINATION REACTIONS

The proteins essential to homologous recombination in E. coli have bee n purified and their individual activities have been identified, permi tting biochemical reconstitution of steps that comprise the cellular r ecombination process. This review focuses on the biochemical events re sponsible for the initiation and homologous pairing steps of genetic r ecombination. The properties of an in vitro recombination reaction tha t requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(chi), are describ ed. The recBCD enzyme serves as the initiator of this reaction; its DN A helicase activity produces single-stranded DNA that is used by the r ecA protein to promote homologous pairing and DNA strand invasion of s upercoiled (recipient) DNA. The SSB protein acts to trap the single-st randed DNA produced by recBCD enzyme and to facilitate pairing by the recA protein. The chi regulatory sequence acts in cis by attenuating t he nuclease, but not the helicase, activity of recBCD enzyme. This att enuation assures the preservation of ssDNA produced by the DNA helicas e activity and is responsible for the simulation in vitro and, presuma bly, in vivo. The attenuation of nuclease activity by chi results in t he loss or functional inactivation of the recD subunit.