Cofactor induced dissociation of the multifunctional multisubunit EcoR124I investigated using electromobility shift assays, AFM and SPR

Abstract

We have applied three techniques to the study of subunit assembly of the Type IC Restriction–Modification enzyme EcoR124I. This fully functional enzyme EcoR124I consists of a complex of the three subunits HsdR, HsdM and HsdS in a R₂M₂S₁ stoichiometry, but is known to dissociate readily, releasing free HsdR and producing first an R₁-complex and then the core, DNA-binding methyltransferase (M₂S₁) complex. Analysis of the assembly pathway of this enzyme has previously employed gel retardation and Surface Plasmon Resonance (SPR), but the studies to date have not included the cofactors required for full enzyme activity. In this paper, we have also used atomic force microscopy (AFM)-based molecular volume measurements, and have analysed the effect of the cofactors ATP and AdoMet on enzyme stability and subunit assembly. We compare the data obtained from all three techniques and we show that they all give consistent results, but inherent differences in the methodologies provide additional information useful for the study of subunit assembly.