ANALYSIS OF THE SUBUNIT ASSEMBLY OF THE TYPE-IC RESTRICTION-MODIFICATION ENZYME ECOR124I

Type I restriction-modification (R-M) enzymes are composed of three di fferent subunits, of which HsdS determines DNA specificity, HsdM is re sponsible for DNA methylation and HsdR is required for restriction. Th e HsdM and HsdS subunits can also form an independent DNA methyltransf erase with a subunit stoichiometry of M2S1, We found that the purified EcoR1241 R-M enzyme was a mixture of two species as detected by the p resence of two differently migrating specific DNA-protein complexes in a gel retardation assay. An analysis of protein subunits isolated fro m the complexes indicated that the larger species had a stoichiometry of R2M2S1 and the smaller species had a stoichiometry of R1M2S1 In vit ro analysis of subunit assembly revealed that while binding of the fir st HsdR subunit to the M2S1 complex was very tight, the second HsdR su bunit was bound weakly and it dissociated from the R1M2S1 complex with an apparent K-d of similar to 2.4 x 10(-7) M. Functional assays have shown that only the R2M2S1 complex is capable of DNA cleavage, however , the R1M2S1 complex retains ATPase activity. The relevance of this si tuation is discussed in terms of the regulation of restriction activit y in vivo upon conjugative transfer of a plasmid-born R-M system into an unmodified host cell.