Puri®cation and characterisation of a novel DNA methyltransferase, M.AhdI

We have cloned the M and S genes of the restrictionmodi®cation (R-M) system AhdI and have puri®ed the resulting methyltransferase to homogeneity. M.AhdI is found to form a 170 kDa tetrameric enzyme having a subunit stoichiometry M2S2 (where the M and S subunits are responsible for methylation and DNA sequence speci®city, respectively). Sedimentation equilibrium experiments show that the tetrameric enzyme dissociates to form a heterodimer at low concentration, with Kd » 2 mM. The intact (tetrameric) enzyme binds speci®cally to a 30 bp DNA duplex containing the AhdI recognition sequence GACN5GTC with high af®nity (Kd » 50 nM), but at low enzyme concentration the DNA binding activity is governed by the dissociation of the tetramer into dimers, leading to a sigmoidal DNA binding curve. In contrast, only non-speci®c binding is observed if the duplex lacks the recognition sequence. Methylation activity of the puri®ed enzyme was assessed by its ability to prevent restriction by the cognate endonuclease. The subunit structure of the M.AhdI methyltransferase resembles that of type I MTases, in contrast to the R.AhdI endonuclease which is typical of type II systems. AhdI appears to be a novel R-M system with properties intermediate between simple type II systems and more complex type I systems, and may represent an intermediate in the evolution of R-M