Mode of action on EcoRI restriction endonuclease: EcoRI and EcoRI variant N199H have active monomeric forms

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Mode of action on EcoRI restriction endonuclease: EcoRI and EcoRI variant N199H have active monomeric forms
Jae Jong Kim; Suk Hoon Koh; Joong Su Kim; Dae Sil Lee
Bibliographic Citation
BMB Reports, vol. 31, no. 2, pp. 149-155
Publication Year
The N199H variant of the EcoRI endonuclease has about twice the catalytic activity of the wild-type. A comparison of their biochemical characteristics, using synthetic oligonucleotides 5′-dAAAACTTAAGAA-AAAAAAAAA-3′ (KA) and 5′-dTTTTTGAATTCT-TTTTTTTTT-3′ (KT), helps to define the cleavage reaction pathway of these enzymes. Both EcoRI and EcoRI variant N199H were found to cleave single-stranded KA or KT about three times faster than the double-stranded forms, although the KT oligonucleotide was more susceptible. Using the ssDNA substrate in kinetic analyses, lower Km values were obtained for the N199H variant than for the wild-type at low (50 mM), as well as high (200 mM), sodium chloride concentrations. This difference between the endonucleases is attributed to a greater accessibility for the substrate by the variant, and also a higher affinity for the DNA backbone. It also appears that the relative activities of the two enzymes, particularly at high ionic strength, are proportional to their populations in the monomeric enzyme form. That is, according to gel filtration data, half of the N199H molecules exist as monomers in 200 mM NaCl, whereas those of the wild-type are mainly dimeric. Consequently, the Asp199 residue of the EcoRI endonuclease may be implicated in the protein-protein interaction leading to dimerization, as well as in coupling to DNA substrates. In summary, it is proposed that active monomeric endonuclease molecules, derived from the dimeric enzyme, recognize and form a complex with a single stranded form of the DNA substrate, which then undergoes nuclcophilic substitution and cleavage.
EcoRIEcoRI variant N199HMode of actionMonomeric form
Korea Soc-Assoc-Inst
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Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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