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The McrBC restriction endonuclease assembles into a ring structure in the presence of G nucleotides
Author(s) -
Panne Daniel,
Müller Shirley A.,
Wirtz Sabine,
Engel Andreas,
Bickle Thomas A.
Publication year - 2001
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/20.12.3210
Subject(s) - basel i , biology , basel ii , economics , microeconomics , capital requirement , incentive
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, 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 endonuclease. McrB S 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, McrB L and McrB S form high molecular weight oligomers in the presence of Mg 2+ 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 McrB L and McrB S revealed ring‐shaped particles with a central channel. Mass analysis by scanning transmission electron microscopy indicates that McrB L and McrB S 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.