Cloning and Engineering of Nicking enzymes

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DESCRIPTION (provided by applicant): This proposal is aimed towards cloning, expression and engineering of DNA nicking enzymes. Two nicking enzymes CviNY2A and CviNYSI have been found in Chlorella-like algae virus strains NY2A and NYSI with three-nucleotide recognition sequence. The cloning and expression of these two enzymes will facilitate application of nicking enzymes in detection of genetic alterations and recombinant DNA technology. Sapl (GCTCTTC NIlN4) is one of the few type IIS endonucleases that have 7-bp recognition sequences. The second goal is to engineer the Sapl restriction endonuclease to isolate variants that nick DNA. There are only seven nicking enzymes are currently commercially available. Due to the difficulty in making large amount of lysates, CviNY2A and CviNYSI nicking enzymes are only available in small quantity and are often in short supply. Therefore CviNY2 and CviNYSI are in urgent need to be cloned and over-expressed to meet the demand in the research community. The following applications may employ the use of nicking enzymes: DNA amplification, recombinant DNA technology for gene assembly, genetic polymorphism detection, specific genome targeting, preparation of nicked duplex DNA for studying DNA mismatch excision repair. Four cloning strategies will be used to clone CviNY2A and CviNYSI: methylase selection of M.CviNY2A and M.CviNYSI and then cloning of the adjacent ORFs, "endo-blue" method for direct cloning of CviNY2A and CviNYSI nicking enzymes, using antibodies to N6A or C5 methylase modified DNA, PCR using degenerate PCR primers based on the conserved amino acid residues of N6A or C5 methylases. Four experimental strategies will be used to engineer Sapl endonuclease and isolate variants that nick DNA: genetic selection by transformation of I exonuclease treated Sapl nicked-circular DNA to enrich plasmids encoding Sapl nicking activity, molecular break lights generated by Sapl nicking enzyme using a high throughput screening system, DNA mobility shift assay, and in vivo SOS induction using a dinD: lacZ indicator strain.