Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites.
EcoRII is unusual among type II restriction enzymes in that, while it cleaves substrates such as pBR322 and bacteriophage lambda that contain several recognition sites for the enzyme efficiently, substrates such as the genomes of bacteriophages T3 and T7 which contain a small number of recognition sites are cut poorly by it. Interestingly, pBR322, or a short DNA duplex containing a single site for the enzyme, can activate the enzyme to cleave resistant substrates. We show here that, at low concentrations, activator short duplexes are themselves cleaved poorly by the enzyme. Further, the reaction shows substrate cooperativity, and at high concentrations, the duplexes are both activators and good substrates for the enzyme. This supports the model that the activation of EcoRII involves binding of more than one DNA molecule and provides a simple system to study the mechanism of activation. Using a gel mobility shift assay, we show that the enzyme forms sequence-specific, methylation-sensitive complexes with the duplexes in the absence of activating DNA. Therefore, resistance of the short duplexes to the enzyme at low concentrations cannot be due to an inability of the enzyme to bind the duplexes. Interestingly, these complexes are stable in the presence of Mg2+, the cofactor for the enzyme, and the complexes obtained in the presence of Mg2+ do not contain DNA that is cleaved by the enzyme. The inefficient step in the action of EcoRII on resistant substrates must occur subsequent to initial substrate binding and it is this step that the activating DNA must regulate.
Gabbara
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Department of Chemistry, Wayne State University, Detroit, Michigan 48202.
Bhagwat
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AS
D001483:Base Sequence; D002413:Cations, Divalent; D004279:DNA, Viral / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D013379:Substrate Specificity; D013604:T-Phages / Q000378:metabolism
D002413:Cations, Divalent; D004279:DNA, Viral; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium