| Abstract [eng] |
Restriction endonucleases are among the most commonly used enzymes in molecular biology. Consequently, changing their very high sequence specificity is one of the scientific goals in studying these enzymes. After unseccessful efforts to generate new specificities by directed mutagenesis it is widely believable that random mutagenesis or its combination with directed mutagenesis can help to generate restriction endonucleases with the new specificity. In addition, random mutagenesis must be combined with effective in vivo selection system. We reasoned that combination of the RM gene complex–mediated postsegregational cell killing (PSK) effect and methylation of overlapping sequences could be used in the selection of active Eco31I restriction endonuclease mutants. To support this idea, eco31IR (recognition sequence 5‘-GGTCTC-3‘) after the random mutagenesis was cloned into stable vector pUC19 (carrying eco31IM gene) and alw26IM (5‘-GTCTC-3‘ recognition sequence) gene was cloned into thermosensitive (ts) vector. Upon loss of ts plasmid at high temperature, the SOS response was induced by eco31IR gene–mediated PSK effect. Thus, transformants carrying a changed specificity Eco31I restriction endonuclease could be selected by indicative blue colour of colony–forming cells in case if special reporter E.coli strain is used. We succeeded in isolating of 8 active Eco31I mutants. Six of them had common D231V amino acid substitution resulting in a weaking of enzyme activity with the change of specificity. Supposedly, Q69R/K127I and E71V mutations were found to be responsible for the changed specificity of others two mutants. The most active of them Eco31IQ69R/K127I preferred to cut its canonical 5‘-GGTCTC-3‘ target and also cleaved 5‘-AGTCTC-3‘, 5‘-TGTCTC-3‘ sequences. Thus, it was identified Eco31IQ69R/K127I specificity - 5’-(G/W)GTCTC-3’. These results allow to suppose that the approach we used in this work could be successfully used in all experiments were catalytically active mutants of restriction enzymes are expected to be isolated. |
