The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

The RecBCD enzyme is required for homologous recombination and DNA repair i n Escherichia coil. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5'-GCTGGTGC-3'). It has been hypothesized that the RecD subunit plays a role in Chi-dependent r egulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rose nberg. S. M., stahl. F. W. & Stahl, M. (1988) in Mechanisms and Consequence s of DNA Damage Processing, eds. Friedberg, E. & Hanawalt. P. (Liss, New Yo rk), pp. 413-422; Churchill, J. J., Anderson. D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901-911]. We tested the hypothesis that the RecD sub unit inhibits recombination by deleting recD from the nuclease- and recombi nation-deficient mutant recB(D1080)ACD. We report here that the resulting s train, recB(D1080A)C. was proficient for recombination and DNA repair. Reco mbination proficiency was accompanied by a change in enzyme activity: RecB( D1080A)C enzyme loaded RecA protein onto DNA during DNA unwinding whereas R ecB(D1080A)CD enzyme did not. Together, these genetic and biochemical resul ts demonstrate that RecA loading by RecBCD enzyme is required for recombina tion in E. coli cells and suggest that RecD interferes with the enzyme doma in required for its loading. A nuclease-dependent signal appears to be requ ired for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the e nzyme acts at Chi.