A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protei n interactions. The most important protein-protein interactions are. those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M2S1. Here, w e analysed the impact on the restriction and modification activities of the change Trp(212) --> Arg in the distal border of the central conserved regi on of the EcoR124I HsdS subunit. We demonstrate that this point mutation si gnificantly influences the ability of the mutant HsdS subunit to assemble w ith the HsdM subunit to produce a functional MTase. As a consequence of thi s, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, H sdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentra tions in vitro of HsdR, restoring restriction activity. (C) 2000 Academic P ress.