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McrBC: a multisubunit GTP-dependent restriction endonuclease
E Sutherland1, L Coe, E A Raleigh
1New England Biolabs, Beverly, MA 10915.
Abstract:
McrBC-mediated restriction of modified DNA has been studied extensively by genetic methods, but little is known of its molecular action. We have used overproducing plasmid constructs to facilitate purification of the McrBL and McrC proteins, and report preliminary characterization of the activity of the complex. Both proteins are required for cleavage of appropriately modified DNA in vitro, in a reaction absolutely dependent on GTP. ATP inhibits the reaction. The sequence and modification requirements for cleavage of the substrate reflect those seen in vivo. The position of cleavage was examined at the nucleotide level, revealing that cleavage occurs at multiple positions in a small region. Based upon these observations, and upon cleavage of model oligonucleotide substrates, it is proposed that the recognition site for this enzyme consists of the motif RmC(N40-80)RmC, with cleavage occurring at multiple positions on both strands, between the modified C residues. In subunit composition, cofactor requirement, and relation between cleavage and recognition site, McrBC does not fit into any of the classes (types I to IV) of restriction enzyme so far described.
Insights
The McrBC enzyme complex cleaves modified DNA using GTP, with ATP inhibiting the process. Its recognition site is proposed as RmC(N40-80)RmC, with cleavage occurring between modified cytosines.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- McrBC-mediated DNA restriction is genetically well-studied, but its molecular mechanisms remain unclear.
- Understanding McrBC's action is crucial for deciphering DNA modification and restriction systems.
Purpose of the Study:
- To characterize the molecular action of the McrBC enzyme complex.
- To determine the in vitro cleavage activity and substrate specificity of purified McrBC.
Main Methods:
- Purification of McrBC proteins using overproducing plasmid constructs.
- In vitro cleavage assays using modified DNA substrates and model oligonucleotides.
- Nucleotide-level analysis of DNA cleavage positions.
Main Results:
- McrBC requires both McrBL and McrC subunits for DNA cleavage in vitro.
- Cleavage is dependent on GTP and inhibited by ATP.
- The recognition site is proposed as RmC(N40-80)RmC, with cleavage occurring at multiple positions between modified cytosines.
- Cleavage patterns in vitro mirror in vivo observations.
Conclusions:
- McrBC functions as a GTP-dependent DNAse with a specific recognition motif.
- The enzyme's mechanism and properties distinguish it from known restriction enzyme classes.
- This study provides a molecular basis for understanding McrBC-mediated DNA restriction.