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 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-gamma-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.
Panne
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Department of Microbiology and Maurice E.Müller Institute for Structural Biology, Biozentrum, Basel University, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Müller
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SA
Wirtz
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Engel
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A
Bickle
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TA
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008855:Microscopy, Electron, Scanning / Q000379:methods; D020836:Protein Structure, Quaternary
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; C069326:mcrC protein, E coli; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D004262:DNA Restriction Enzymes; C120301:McrBC endonuclease; C059678:mcrB protein, E coli