Purification and biochemical characterisation of the EcoR124 type I modification methylase

Abstract

Large scale purification of the type I modification methylase EcoR124 hias been achieved from an over-expressing strain by a two step procedure using ion-exchange and heparin chromatography. Pure methylase is obtained at a yield of 30mg per gm of cell paste. Measurements of the molecular weight and subunit stoichiometry show that the enzyme is a trimeric complex of 162 kDa consisting of two subunits of HsdM (58 kDa) and one subunit of HsdS (46 kDa). The purified enzyme can methylate a DNA fragment bearing its cognate recognition sequence. Binding of the methylase to synthetic DNA fragments containing either the EcoR124 recognition sequence GAAN6RT-CG, or the recognition sequence GAAN7RTCG of the related enzyme EcoR124/3, was followed by fluorescence competition assays and by gel retardation analysis. The results show that the methylase binds to its correct sequence with an affinity of the order 108 M-1 forming a 1: 1 complex with the DMA, The affinity for the incorrect sequence, differing by an additional base pair in the non-specific spacer, is almost two orders of magnitude lower. © 1992 Oxford University Press.