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The bcgl restriction endonuclease acting currently at eight phosphodiester bonds

Reference	BB/C513077/1
Principal Investigator / Supervisor	Professor Steve Halford
Co-Investigators / Co-Supervisors
Institution	University of Bristol
Department	Biochemistry
Funding type	Research
Value (£)	380,503
Status	Completed
Type	Research Grant
Start date	05/05/2005
End date	04/05/2010
Duration	60 months

Abstract

The Bcgl restriction endonuclease exemplifies a group of restriction enzymes, known as the Type IIB enzymes, that recognise bipartite DNA sequences and which cut both strands of the DNA on both sides of the recognition sequence. It excises a 34 bp fragment that contains the recognition site from the remainder of the DNA. Moreover, both Bcgl and almost all of the other restriction enzymes that act in this manner have to interact with two copies of their recognition sequence before they can cut DNA. They generally convert DNA with two sites directly to the products cut at both loci, without releasing intermediates cut at just one locus. They can thus cleave eight phosphodiester bonds in one turnover. Moreover, each turnover also incorporates four methyltransferase reactions: endonuclease action requires two unmethylated sites but the 34 bp products come out methylated in both strands. We plan to first determine the subunit organisation of Bcgl by gel filtration and analytical ultracentrifugation, for both the free protein and its complexes with specific oligoduplexes. Bcgl contains two sorts of subunits, A and B, probably (but not definitively) in the ratio A2B1: other Type IIB enzymes may have an A1B1 composition or may even contain just a single polypeptide. The assembly state is unknown at present but to carry out 12 distinct reactions at separate DNA sites, it seems likely that it acts on DNA as a large multimeric assembly. The mode of action of the enzyme will then be elucidated by examining the kinetics of its reactions on plasmids with one and with two copies of the recognition sequence, on DNA catenanes and on oligoduplex substrates. The cleavage of the individual phosphodiester bonds, either side of the recognition site, will be monitored in order to reveal whether these are cut in sequential or parallel reactions: likewise the methylation of each strand. Phosphorothioate substrates will be used to reveal whether the active site(s) act independently at each phosphodiester bond, or whether they move from one bond to the next. The assembly of the synaptic complexes will be analysed initially by equilibrium binding methods, and subsequently by direct methods from single molecule mechanics. Enzymes that recognise bipartite DNA sequences often employ discrete protein domains to contact the separate elements of their recognition sequences. In some cases, the re-assortment of domains amongst related proteins has produced enzymes with novel sequence specificities: for example, by combining from two proteins that recognise two different bipartite sequences the domain that contacts the first part of one sequence with the domain that contacts the second part of the alternative sequence. But the generation of a new sequence specificity has yet to be achieved by any Type II restriction enzyme, the enzymes that are universally used as tools for the analysis and manipulation of DNA.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Industrial Biotechnology, Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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