PMID:7781618
| Citation |
Krüger, T, Wild, C and Noyer-Weidner, M (1995) McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues. EMBO J. 14:2661-9 |
|---|---|
| Abstract |
Restriction of DNA by the Escherichia coli K-12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation. From previous work by others it was known that restriction of 5-methylcytosine-containing DNA requires two methylated 5'-PuC sites separated by approximately 40-80 non-defined base pairs. Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit. McrB has a low affinity for non-methylated DNA, with which it forms low molecular weight complexes. The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi- or fully methylated 5'-PuC sites. Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules. Methylation at unique 5'-PuC sites can be sufficient to stimulate DNA binding by McrB. As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5'-PumC sites. The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non-sequence-specific manner with DNA containing methylated 5'-CpG sequences and might point to a common molecular origin of these proteins. In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC. McrC neither binds to DNA nor modulates the DNA binding potential of McrB. As McrC is essential for restriction it appears to predominantly function in catalysis. |
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| Keywords |
Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Binding Sites; Cytosine/chemistry; Cytosine/metabolism; DNA Restriction Enzymes/metabolism; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Protein Binding; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Substrate Specificity |
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