Abstract: A novel restriction endonuclease and methods of making the same are obtainable from either Citrobacter species 2144 (NEB#1398) or the recombinant stain Escherichia coli (NEB#1554) which cleaves at nt sequence 5?-CAANNNNNGTGG-3? (SEQ ID NO:14) in double-stranded DNA molecules. The novel restriction endonuclease is a modular protein in which the specificity moiety is an independent module from the restriction-modification module.

Abstract: Methods are provided for converting into a sequence specific strand specific and location specific DNA nicking endonuclease, a restriction endonuclease that recognizes an asymmetric DNA sequence, the endonuclease having two catalytic sites and one or more single sequence specific DNA-binding domains. In one embodiment the method requires inactivating one of the catalytic sites of the restriction endonuclease. In another embodiment, the restriction endonuclease is a dimer having a first and second subunit each comprising a sequence specific DNA binding domain, a catalytic site and a dimerization domain. The nicking endonuclease is formed from combining one subunit having an inactivated catalytic site and a second subunit having an inactivated DNA binding domain. The nicking endonuclease may be converted into a restriction endonuclease by the addition of manganese cations in the digestion buffer.

Abstract: Methods are provided for converting into a sequence specific strand specific and location specific DNA nicking endonuclease, a restriction endonuclease that recognizes an asymmetric DNA sequence, the endonuclease having two catalytic sites and one or more single sequence specific DNA-binding domains. In one embodiment the method requires inactivating one of the catalytic sites of the restriction endonuclease. In another embodiment, the restriction endonuclease is a dimer having a first and second subunit each comprising a sequence specific DNA binding domain, a catalytic site and a dimerization domain. The nicking endonuclease is formed from combining one subunit having an inactivated catalytic site and a second subunit having an inactivated DNA binding domain. The nicking endonuclease may be converted into a restriction endonuclease by the addition of manganese cations in the digestion buffer.