INTERACTION WITH THE RECOMBINATION HOT-SPOT CHI IN-VIVO CONVERTS THE RECBCD ENZYME OF ESCHERICHIA-COLI INTO A CHI-INDEPENDENT RECOMBINASE BY INACTIVATION OF THE RECD SUBUNIT

The RecBCD enzyme of Escherichia coli promotes recombination preferent ially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing ph age lambda red gam crosses, phage T4 2(-) survival measurements, and e xoV assays, it is shown that E. coli cells in which RecBCD has extensi ve opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-indu ced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximate to 2 h. It is conclud ed that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is rel eased and initiates recombination on other DNA molecules in a chi-inde pendent fashion. Overexpression of recD(+) (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi -stimulated recombination, The observed recA(+) dependence of the down regulation of exoV could explain the previously noted ''reckless'' DNA degradation of recA mutants,It is proposed that chi sites are regulat ory elements for the RecBCD to RecBC switch and thereby function as ci s- and trans-acting stimulators of RecBC-dependent recombination.