Restriction endonucleases are essential tools in genetic engineering, DNA cloning and DNA diagnostics. The nicking endonuclease N.BstNBI, anew type of endonuclease, is the only commercially available endonuclease which introduces a nick at a specific sequence. The unique nicking activity is useful in DNA manipulation and this research plan proposes a way to generate more nicking endonucleases via protein engineering. The type IIs endonuclease P1eI is a good candidate for engineering. P1eI recognizes exactly the same sequence as N.BstNbI, but it breaks both DNA strands like most restriction enzymes. The genes encoding N.BstNBI and PleI endonucleases will be cloned and sequenced, Mutagenesis will be performed to identify the catalytic sites of cleavage for N.BstNBI and PleI. In addition, biochemical assays will be performed to determine the cleavage activities of both enzymes. We ill compare the protein sequences, the catalytic sites and biochemical characteristics of both enzymes. Once cleavage mechanisms and the differences of N.BstNBI and PleI in cleavage reactions are revealed, the double strand cleavage activity of PleI will be altered to mimic the nicking activity of N.BstNBI. If this research succeeds, the same technique can be used to convert more type IIs enzymes into nicking enzymes.
Potentially can lead to the conversion of type IIs restriction endonucleases into nicking enzymes which have great applications in DNA amplification technology (SDA, RCA) and research (DNA replication, repair, etc.).
