DNA polymerase mutant having one or more mutations in the active site

• US 20020132249A1
• Filed: 10/16/2001
• Published: 09/19/2002
• Est. Priority Date: 01/14/2000
• Status: Active Grant

• Current Assignee: Lawrence A. Loeb, Premal H. Patel
• Original Assignee: Lawrence A. Loeb, Premal H. Patel
• Inventors: Patel, Premal H., Loeb, Lawrence A.
• Granted Patent: US 6,602,695 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

This invention provides a DNA polymerase that is a mutant form of a naturally occurring DNA polymerase, of which one or more amino acids in the active site are mutated. The DNA polymerase mutant of this invention is characterized by altered fidelity or altered enzymatic activity in comparison with the naturally occurring DNA polymerase. For example, the DNA polymerase mutant provides increased enzymatic activity, altered dNTP/rNTP specificity, or enhanced fidelity. In one aspect of the invention, the naturally occurring DNA polymerase comprises an amino acid sequence motif: AspTyrSerGlnIleGluLeuArg in the active site. In another aspect of the invention, the naturally occurring DNA polymerase comprises an amino acid sequence motif: LeuLeuValAlaLeuAspTyrSerGlnIleGluLeuArg in the active site. The mutant DNA polymerase has been altered in the active site of the naturally occurring DNA polymerase to contain either (a) two or more amino acid substitutions in the amino acid sequence motif, or (b) a substitution of an amino acid other than Glu in the amino acid sequence motif.

Claims

1. A mutant DNA polymerase within the Pol I family of polymerases, comprising a mutation in an active site of a naturally occurring DNA polymerase, wherein said active site comprises an amino acid sequence LeuLeuValAlaLeuAspTyrSerGlnIleGluLeuArg (SEQ ID NO:

3), said mutation comprises an alteration of an amino acid other than Glu in said sequence, and said mutant DNA polymerase possesses altered fidelity or altered catalytic activity in comparison with said naturally occurring DNA polymerase.

2. The mutant DNA polymerase according to claim 1, wherein said Asp of said amino acid sequence motif is not altered in said mutant form.

3. The mutant DNA polymerase according to claim 1, wherein said mutant DNA polymerase incorporates a ribonucleotide at a rate at least 10 fold greater than that of said naturally occurring DNA polymerase.

4. The mutant DNA polymerase according to claim 3, wherein said mutation comprises an alteration of Ile in said amino acid sequence.

5. The mutant DNA polymerase according to claim 4, wherein said Ile is altered to a hydrophilic amino acid in said mutant form.

6. The mutant DNA polymerase according to claim 3, wherein said mutation comprises two or more amino acid substitution in said amino acid sequence.

7. The mutant DNA polymerase according to claim 3, wherein said mutant DNA polymerase functions as both DNA polymerase and RNA polymerase.

8. The mutant DNA polymerase according to claim 1, wherein said polymerase incorporates an unconventional nucleotide at a rate at least 10 fold greater than that of said naturally occurring DNA polymerase.

9. The mutant DNA polymerase according to claim 8, wherein said unconventional nucleotide is a ribonucleotide analog.

10. The mutant DNA polymerase according to claim 8, wherein said unconventional nucleotide comprises a base labeled with a reporter molecule.

11. The mutant DNA polymerase according to claim 10, wherein said reporter molecule is a fluorophore or a hapten.

12. The mutant DNA polymerase according to claim 8, wherein said unconventional nucleotide is a chemotherapy drug.

13. The mutant DNA polymerase according to claim 12, wherein said chemotherapy drug is ara-C or acyclovir.

14. The mutant DNA polymerase according to claim 8, wherein said unconventional nucleotide is an anti-viral or an anti-cancer drug.

15. The mutant DNA polymerase according to claim 1, which has an increased catalytic efficiency for incorporating deoxyribonucleotide over said naturally occurring DNA polymerase.

16. The mutant DNA polymerase according to claim 1, wherein said naturally occurring DNA polymerase is a thermostable Thermus species DNA polymerase.

17. The mutant DNA polymerase according to claim 16, wherein said Thermus species is Thermus aqaticus.

18. The mutant DNA polymerase according to claim 1, which possesses enhanced fidelity comparing with said naturally occurring DNA polymerase.

19. An isolated nucleic acid sequence encoding the mutant DNA polymerase according to claim 1.

20. A mutant DNA polymerase within the Pol I family of polymerases, comprising a mutation in an active site of a naturally occurring DNA polymerase, wherein said active site comprises an amino acid sequence LeuLeuValAlaLeuAspTyrSerGlnIleGluLeuArg (SEQ ID NO:

3), said mutation comprises two or more amino acid substitutions, and said mutant DNA polymerase possesses altered fidelity or altered catalytic activity in comparison with said naturally occurring DNA polymerase.

21. The mutant DNA polymerase according to claim 20, wherein said Asp of said amino acid sequence motif is not altered in said mutant form.

22. The mutant DNA polymerase according to claim 20, wherein said mutant DNA polymerase incorporates a ribonucleotide at a rate at least 10 fold greater than that of said naturally occurring DNA polymerase.

23. The mutant DNA polymerase according to claim 22, wherein said mutation comprises an alteration of Ile in said amino acid sequence.

24. The mutant DNA polymerase according to claim 23, wherein said Ile is altered to a hydrophilic amino acid in said mutant form.

25. The mutant DNA polymerase according to claim 22, wherein said mutant DNA polymerase functions as both DNA polymerase and RNA polymerase.

26. The mutant DNA polymerase according to claim 20, wherein said polymerase incorporates an unconventional nucleotide at a rate at least 10 fold greater than that of said naturally occurring DNA polymerase.

27. The mutant DNA polymerase according to claim 26, wherein said unconventional nucleotide is a ribonucleotide analog.

28. The mutant DNA polymerase according to claim 26, wherein said unconventional nucleotide comprises a base labeled with a reporter molecule.

29. The mutant DNA polymerase according to claim 28, wherein said reporter molecule is a fluorophore or a hapten.

30. The mutant DNA polymerase according to claim 26, wherein said unconventional nucleotide is a chemotherapy drug.

31. The mutant DNA polymerase according to claim 30, wherein said chemotherapy drug is ara-C or acyclovir.

32. The mutant DNA polymerase according to claim 26, wherein said unconventional nucleotide is an anti-viral drug or an anti-cancer drug.

33. The mutant DNA polymerase according to claim 20, which has an increased catalytic efficiency for incorporating deoxyribonucleotide over said naturally occurring DNA polymerase.

34. The mutant DNA polymerase according to claim 20, wherein said naturally occurring DNA polymerase is a thermostable Thermus species DNA polymerase.

35. The mutant DNA polymerase according to claim 34, wherein said Thermus species is Thermus aqaticus.

36. The mutant DNA polymerase according to claim 20, which possesses enhanced fidelity comparing with said naturally occurring DNA polymerase.

37. An isolated nucleic acid sequence encoding the mutant DNA polymerase according to claim 20.