Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleases EcoKI and EcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described. Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts of E. coli. For the first time all three subunits of both EcoKI and EcoR124I were detected as distinct spots on a single 2-D gel. A sensitive immunoblotting procedure was suggested suitable for routine use in determining the identity of individual subunits. Potential application of this method for detailed studies of regulation of the function and stoichiometry of the enzyme complexes is discussed.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/BF02818664 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Structure and operation of the DNA-translocating type I DNA restriction enzymes.
Genes Dev
January 2012
EaStCHEM School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, United Kingdom
Article Synopsis
- Type I DNA restriction/modification enzymes are crucial molecular machines in most bacteria, playing a key role in genetic engineering by managing the uptake of foreign DNA while modifying host DNA for protection.
- These enzymes, like EcoKI and EcoR124I, undergo significant structural changes during function, transitioning from an open to a compact form when they bind DNA.
- The research reveals these enzymes' structures and provides insights into the evolutionary connection between Type I and Type II restriction/modification enzymes through advanced imaging and molecular modeling techniques.
Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes.
Nucleic Acids Res
September 2011
School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.
The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e.
View Article and Find Full Text PDFAn investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme.
Nucleic Acids Res
September 2011
University of Edinburgh, Edinburgh, EH9 3JJ, UK.
Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation.
View Article and Find Full Text PDFPhosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit.
FEMS Microbiol Lett
May 2007
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated.
View Article and Find Full Text PDFControl of the endonuclease activity of type I restriction-modification systems is required to maintain chromosome integrity following homologous recombination.
Mol Microbiol
May 2006
Institute of Structural and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.
A type I restriction-modification enzyme will bind to an unmethylated target sequence in DNA and, while still bound to the target, translocate DNA through the protein complex in both directions. DNA breakage occurs when two translocating complexes collide. However, if type I restriction-modification systems bind to unmodified target sequences within the resident bacterial chromosome, as opposed to incoming 'foreign' DNA, their activity is curtailed; a process known as restriction alleviation (RA).
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!