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Characterization of anEcoR 124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit, which results in a novel DNA specificity

Abstract

We purified and characterized both the methyltransferase and the endonuclease containing the HsdSΔ50 subunit (type I restriction endonucleases are composed of three subunits — HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutationhsdSΔ50 of the type IC R-M systemEcoR124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsdSΔ50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificityin vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme — a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTaseΔ50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused thehsdM andhsdSΔ50 genes and showed that the HsdM-HsdSΔ50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.

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Correspondence to K. Firman.

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The first author was employed on aWellcome Trust Project grant and the third author was employed on aWellcome Trust International Fellowship.

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Abadjieva, A., Scarlett, G., Janščák, P. et al. Characterization of anEcoR 124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit, which results in a novel DNA specificity. Folia Microbiol 48, 319–328 (2003). https://doi.org/10.1007/BF02931361

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  • DOI: https://doi.org/10.1007/BF02931361

Keywords

  • Methylation Status
  • Deletion Mutant
  • Partial Proteolysis
  • Mung Bean Nuclease
  • Active MTase