A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway.
The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.
Weiserova
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School of Biological Sciences, Biophysics Laboratories University of Portsmouth, St. Michael's Building, Portsmouth, PO1 2DT, United Kingdom.
Dutta
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CF
Firman
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D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D008954:Models, Biological; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits
D004268:DNA-Binding Proteins; D021122:Protein Subunits; D004247:DNA; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific