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

AUTOR(ES)

Panne, Daniel

FONTE

Oxford University Press

RESUMO

McrBC from Escherichia coli K-12 is a restriction enzyme that belongs to the family of AAA+ proteins and cuts DNA containing modified cytosines. Two proteins are expressed from the mcrB gene: a full-length version, McrBL, and a short version, McrBS. McrBL binds specifically to the methylated recognition site and is, therefore, the DNA-binding moiety of the McrBC endonuclease. McrBS is devoid of DNA-binding activity. We observed that the quaternary structure of the endonuclease depends on binding of the cofactors. In gel filtration experiments, McrBL and McrBS form high molecular weight oligomers in the presence of Mg2+ and GTP, GDP or GTP-γ-S. Oligomerization did not require the presence of DNA and was independent of GTP hydrolysis. Electron micrographs of negatively stained McrBL and McrBS revealed ring-shaped particles with a central channel. Mass analysis by scanning transmission electron microscopy indicates that McrBL and McrBS form single heptameric rings as well as tetradecamers. In the presence of McrC, a subunit that is essential for DNA cleavage, the tetradecameric species was the major form of the endonuclease.

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