MAPPING OF METAL ION BINDING SITES IN THE RECBCD ENZYME AND THE ROLE OF MAGNESIUM IN THE SUBUNIT INTERACTIONS OF RECBCD

Abstract

E. coli RecBCD is a 330kDa enzyme with three subunits RecB, RecC and RecD. The biological functions of RecBCD are to degrade foreign linear DNA and provide single stranded DNA to initiate homologous recombination. The enzyme has nuclease, helicase and ATPase activities. Divalent metal ions like Mg2+ are required for the nuclease and helicase activity of RecBCD. Previous work done by Dixon et al. (Dixon, D. A., Churchill, J. J., and Kowalczykowski, S. C. (1994) Proc. Natl. Acad. Sci. USA 91, 2980-2984) and Taylor and Smith (Taylor, A. F., and Smith, G. R. (1999) Genes & Devel. 13, 890-900) has shown that Mg2+ plays an important role in the interactions of the subunits and the activities of RecBCD. Identification of metal binding sites and the role of Mg2+ in the interaction of the subunits will enhance our understanding of the catalytic mechanism and structure of E. coliRecBCD.

The RecB subunit contains the 30kDa nuclease domain of RecBCD. Sequence comparisons have shown that the nuclease active site of the 30kDa nuclease domain is very similar to that of restriction endonucleases. Fenton chemistry techniques have been used to map the metal binding sites of several restriction enzymes. We used Fenton chemistry techniques coupled with Edman sequencing to map the Mg2+ metal binding site of the 30kDa nuclease domain. A specific amino acid residue, Asp 1067 was identified as the metal binding site. Further corroboration that Asp 1067 was a unique Mg2+ binding site was obtained by doing mutational studies. Studies were also carried out in presence of other metals like Ca2+ to understand the details of the metal binding site.

The interactions between the RecB and RecC subunits of RecBCD are dependent on the presence of Mg2+ and DNA. We studied the role of magnesium on the binding interactions using surface plasmon resonance (Biacore) and found that Mg2+ enhances the binding interactions between the RecB and RecC subunits. Also, the role of magnesium in the binding of the RecC and 100kDa helicase domain of RecC were studied using ssDNA agarose chromatography, spin columns and gel filtration. Since, the 100kDa helicase domain requires Mg2+ for its helicase activity, we employed Fenton chemistry techniques coupled with mass-spectrometry to show that there is a metal binding site in the 100kDa helicase domain.