Abstract: Methods are provided for engineering novel strand-specific nicking endonucleases by means of an in vivo enrichment of a plasmid library containing a randomly mutagenized restriction endonuclease gene. The plasmids contain adjacent to the gene a cleavable or nickable sequence for cleaving or nicking by the endonuclease product of the gene and a second recognition site for a second endonuclease. The plasmid library is used to transform unmodified host cells. Plasmids from the cultured transformed cells may be analyzed by an in vitro assay for nicking and the nicked plasmids pooled and used to transform host cells. The product is then pooled and the single-stranded specificity of the endonuclease is then determined. The product is either cloned after amplification or identified by use of a selectable marker.

Abstract: The present invention relates to recombinant DNA encoding the AcuI restriction endonuclease as well as AcuI methylase, and expression of AcuI restriction endonuclease and AcuI methylase in E. coli cells containing the recombinant DNA.

Abstract: Methods and compositions are provided for altering the DNA recognition and cleavage characteristics of an endonuclease without prior knowledge of the endonuclease's three-dimensional structure and/or amino acid residues responsible for activity and/or specificity. Methods include subjecting a mutagenized endonuclease gene library to a genetic selection in prokaryotic cells which tolerate the expression of mutated endonuclease and where the endonuclease is active and determining the altered recognition-site specificity for the endonuclease.

Abstract: Methods are provided for engineering novel strand-specific nicking endonucleases by means of an in vivo enrichment of a plasmid library containing a randomly mutagenized restriction endonuclease gene. The plasmids contain adjacent to the gene a cleavable or nickable sequence for cleaving or nicking by the endonuclease product of the gene and a second recognition site for a second endonuclease. The plasmid library is used to transform unmodified host cells. Plasmids from the cultured transformed cells may be analyzed by an in vitro assay for nicking and the nicked plasmids pooled and used to transform host cells. The product is then pooled and the single-stranded specificity of the endonuclease is then determined. The product is either cloned after amplification or identified by use of a selectable marker.

Abstract: Methods are provided for altering the cleavage specificity of a Type IIG restriction endonuclease, the Type IIG restriction endonuclease being characterized by a cleavage domain adjacent to a methylase domain, the methylase domain located adjacent to a specificity domain. The method includes ligating DNA or protein sequences to form a fusion DNA or fusion protein. Where a fusion DNA is formed, the host cell is transformed with the fusion DNA to express a Type IIG restriction endonuclease with altered cleavage specificity.

Abstract: The present invention relates to recombinant DNA encoding the AcuI restriction endonuclease as well as AcuI methylase, and expression of AcuI restriction endonuclease and AcuI methylase in E. coli cells containing the recombinant DNA.

Abstract: The present invention relates to recombinant DNA encoding the PpuMI restriction endonuclease as well as PpuMI methylase, expression of PpuMI restriction endonuclease and PpuMI methylase in E. coli cells containing the recombinant DNA.

Abstract: The present invention relates to recombinant DNA encoding the OkrAI restriction endonuclease as well as OkrAI methylase, expression of OkrAI restriction endonuclease in E. coli cells containing the recombinant DNA.

Abstract: The present invention relates to recombinant DNA encoding the PpuMI restriction endonuclease as well as PpuMI methylase, expression of PpuMI restriction endonuclease and PpuMI methylase in E. coli cells containing the recombinant DNA.

Abstract: The present invention relates to recombinant DNA which encodes the BstYI restriction endonuclease as well as BstYI methyltransferase, expression of BstYI restriction endonuclease and M.BstYI in E. coli cells containing the recombinant DNA. It also relates to methods for purification of the recombinant BstYI restriction endonuclease and BstYI methyltransferase.