The DNA recognition subunit of the type is restriction-modification enzymeEcoAI tolerates circular permutions of its polypeptide chain

The DNA specificity subunit (HsdS) of type I restriction-modification enzym es is composed of two independent target recognition domains and several re gions whose amino acid sequence is conserved within an enzyme family. The c onserved regions participate in intersubunit interactions with two modifica tion subunits (HsdM) and two restriction subunits (HsdR) to form the comple te endonuclease. It has been proposed that the domains of the HsdS subunit have a circular organisation providing the required symmetry for their inte raction with the other subunits and with the bipartite DNA target. To test this model, we circularly permuted the HsdS subunit of the type IB R-M enzy me EcoAI at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circula rly permuted variants of HsdS that had termini located at equivalent positi ons in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity, sugg esting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stab le trimeric complex with HsdM, M2S1, which was fully active as a DNA methyl transferase. Gel electrophoretic mobility shift assays revealed that the Hs dS protein alone was not able to form a specific complex with a 30-mer olig oduplex containing a single EcoAI recognition site. However, addition of st oichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular or,organisation of domains of t he HsdS subunit. in addition, they suggest a possible role of HsdM subunits in the formation of this structure. (C) 1998 Academic Press.