The McrBC restriction endonuclease assembles into a ring structure in the presence of G nucleotides

McrBC from Escherichia coli K-12 is a restriction enzyme that belongs to th e family of AAA(+) proteins and cuts DNA containing modified cytosines, Two proteins are expressed from the mcrB gene: a full-length version, McrB(L), and a short version, McrB(S). McrB(L) binds specifically to the methylated recognition site and is, therefore, the DNA-binding moiety of the McrBC en donuclease, McrB(S) is devoid of DNA-binding activity. We observed that the quaternary structure of the endonuclease depends on binding of the cofacto rs, In gel filtration experiments, McrB(L) and McrB(S) form high molecular weight oligomers in the presence of Mg2+ and GTP, GDP or GTP-gamma -S. Olig omerization did not require the presence of DNA and was independent of GTP hydrolysis. Electron micrographs of negatively stained McrB(L) and McrB(S) revealed ring-shaped particles with a central channel. Mass analysis by sca nning transmission electron microscopy indicates that McrB(L) and McrB(S) f orm single heptameric rings as well as tetradecamers, In the presence of Mc rC, a subunit that is essential for DNA cleavage, the tetradecameric specie s was the major form of the endonuclease.