Claims
- 1. A method 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, the method comprising:
inactivating one of the catalytic sites of the restriction endonuclease by modifying the nucleotide sequence coding for selected amino acid residues in the catalytic site so as to convert the restriction endonuclease into the nicking endonuclease.
- 2. A method according to claim 1, wherein the restriction endonuclease has one single sequence specific DNA-binding domain.
- 3. A method according to claim 1 wherein the restriction endonuclease further comprises a dimerization domain that remains unmodified in the nicking endonuclease.
- 4. A method according to claim 1, wherein the two catalytic sites of the restriction endonuclease comprise different amino acid sequences.
- 5. A method according to claim 1, wherein the two catalytic sites of the restriction endonuclease comprise the same amino acid sequence.
- 6. A method according to claim 2, wherein the restriction endonuclease is selected from BstNB1 and Bbv1.
- 7. A method according to claim 5, wherein the restriction endonuclease is a dimer, the dimer having a first and a second subunit wherein the first and the second subunit each comprise a sequence specific DNA-binding domain, a catalytic site and a dimerization domain, the method further comprising modifying the first subunit by inactivating the catalytic site and modifying the second subunit by inactivating the DNA-binding domain, the modified dimer forming the nicking endonuclease.
- 8. A method according to claim 1, wherein the restriction endonuclease prior to modification is a homodimer and the nicking endonuclease is functionally a heterodimer.
- 9. A method according to claim 7, wherein the restriction endonuclease is a type IIs or type IV restriction endonuclease.
- 10. A method according to claim 7, wherein the restriction endonuclease is FokI.
- 11. A method according to claim 1, wherein inactivating one of the catalytic sites further comprises modifying one or more amino acid residues in a predetermined region of one of the catalytic site by site specific mutagenesis of the nucleic acid encoding the residues.
- 12. A method according to claims 2 or 7, wherein inactivating one of the catalytic sites further comprises: modifying by site specific mutagenesis, a nucleic acid sequences encoding the residues of one or more amino acid residues in a predetermined region of the restriction endonuclease so as to inactivate one of the catalytic sites or the DNA binding domain
- 13. A method according to claims 2 or 7, wherein inactivating one of the catalytic sites further comprises: modifying by site specific mutagenesis, a nucleic acid sequences encoding the residues of one or more amino acid residues in a predetermined region of the restriction endonuclease so as to inactivate one of the catalytic sites or the DNA binding domain.
- 14. A method according to claims 1, 2 or 7, wherein inactivating one of the catalytic sites further comprises modifying one or more amino acids in a conserved motif in the DNA encoding the restriction endonuclease, the conserved motif identified through alignment of nucleic acid sequences of related proteins.
- 15. A method according to claim 14, wherein the conserved motif encodes any of a proline (P), aspartic acid (D), glutamic acid (E) and lysine (K) amino acids in a PD-EXK motif.
- 16. A method according to claim 1, wherein the restriction endonuclease is selected from AciI, BbvCI, Bpu10I, BsrBI, BssSI, BtrI and GdiII.
- 17. A method according to claim 3, wherein the restriction endonuclease is selected from BsmI, BsrI and SimI.
- 18. A method according to claim 3, wherein the restriction endonuclease is selected from BsrDI, BstF5I and BtsI.
- 19. A method according to claim 3, wherein the restriction endonuclease is selected from BstNBI and BbvI.
- 20. A method for converting into a sequence-specific, strand specific and location specific DNA nicking endonuclease, a Type II restriction endonuclease that recognizes an asymmetric DNA sequence, the endonuclease being a dimer having a first and second subunit wherein the first and the second subunit each comprise a sequence specific DNA-binding domain, a catalytic site and a dimerization domain, the method comprising: modifying the first subunit by inactivating the catalytic site and modifying the second subunit by inactivating the DNA-binding domain, the modified dimer forming the nicking endonuclease,
- 21. A nicking endonuclease made according to any of claims 1, 2, 7 and 20.
- 22. A method of introducing site specific nicks into pre-selected strands of a DNA duplex, comprising mixing double stranded DNA with a nicking endonuclease formed according to any of claims 1, 2, 7 and 20.
- 23. A method of claim 22, wherein the double stranded DNA is circular supercoiled DNA.
- 24. A method of converting into a restriction endonuclease, a DNA nicking endonuclease according to claim 21, comprising; adding manganese cation to a digestion buffer containing the nicking endonuclease.
- 25. A method of inhibiting activity of a nicking endonuclease according to claim 21, comprising: adding calcium to the endonuclease in a digestion buffer.
RELATED APPLICATIONS
[0001] This is a Non-Provisional Application of Provisional Application Serial No. 60/314,386 filed on Aug. 23, 2001 herein incorporated by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60314386 |
Aug 2001 |
US |