Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

• US 6,365,355 B1
• Filed: 08/29/2000
• Issued: 04/02/2002
• Est. Priority Date: 03/28/2000
• Status: Expired due to Fees

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: McCutchen-Maloney, Sandra L.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/650,855
• Time in Patent Office: 581 Days
• Field of Search: 435/6, 435/7.1, 530/350, 536/23.2, 536/23.4
• US Class Current: 435/6.14
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12Y 402/99018 DNA-(apurinic or apyrimidinic site)lyase (4.2.99.18)

Abstract

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

Claims

1. A recombinant chimeric protein comprising a DNA mutation binding protein and a nuclease.

2. The chimeric protein of claim 1 wherein said DNA mutation is selected from the group consisting of DNA sequence variability, single base pair mutations, uracil incorporated DNA, point mutations, DNA mismatches, DNA insertions, DNA deletions, DNA transversions, DNA transitions, frameshift mutations and damaged DNA.

3. The chimeric protein of claim 2 wherein said DNA mutation is a DNA mismatch.

4. The chimeric protein of claim 2 wherein said DNA mutation is a DNA insertion.

5. The chimeric protein of claim 1 wherein said nuclease nonspecifcally cuts DNA.

6. The chimeric protein of claim 1 wherein said nuclease cuts DNA at 7-oxoguanine residues or DNA at guanine/adenine residues.

7. The chimeric protein of claim 1 wherein said DNA mutation binding protein is selected from the group consisting of MutS homologue2, xeroderma pigmentosum complementation group A, xeroderma pigmentosum complementation group C, xeroderma pigmentosum complementation group E, Thermus thermophiluis Mut S, thymine DNA glycosylase, Escherechia coli Fpapy-DNA glycosylase, Escherechia coli endonuclease III, Escherechia coli endonuclease IV, T4 endonuclease, Escherechia coli uracil DNA glycosylase, Escherechia coli A/G-specific adenine DNA glycosylase, Escherechia coli Uvr A and Escherechia coli Uvr B.

8. The chimeric protein of claim 1 wherein said nuclease is selected from the group consisting of human excision repair cross-complementing rodent repair deficiency complementation group 4 protein, Serratia marcescens nuclease, Escherechia coli Fpapy-DNA glycosylase;

Escherechia coli endonuclease III;

Escherechia coli endonuclease IV;

T4 endonuclease;

Escherechia coli uracil DNA glycosylase;

Escherechia coli A/G-specific adenine DNA glycosylase, Escherechia coli Uvr B nuclease and Escherechla coil Uvr C nuclease.

9. The chimeric protein of claim 1 further including a linker peptide.

10. The chimeric protein of claim 9 wherein said linker has the amino acid sequence depicted in SEQ ID NO:

17.

11. The protein of claim 7 wherein xeroderma pigmentosum complementation group F has the amino acid sequence depicted in SEQ ID NO:

11.

12. The protein of claim 7 wherein xeroderma pigmentosum complementation group A has the sequence depicted in SEQ ID NO:

7.

13. The protein of claim 7 wherein MutS homologue 2 has the amino acid sequence depicted in SEQ ID NO:

1 or SEQ ID NO;

3.

14. The protein of claim 7 wherein Thermus thermophilus MutS has the amino acid sequence depicted in SEQ ID NO:

15.

15. The protein of claim 7 wherein xeroderma pigmentosum complementation group E has the amino acid sequence depicted in SEQ ID NO:

21.

16. The protein of claim 7 wherein xeroderma pigmentosum complementation group C has the amino acid sequence depicted in SEQ ID NO:

19.

17. The protein of claim 8 wherein human excision repair cross-complementing rodent repair deficiency complementation group 4 protein (XPF) has the amino acid sequence depicted in SEQ ID NO:

11 or SEQ ID NO;

13.

18. The protein of claim 8 wherein Serratia marcescens nuclease has the amino acid sequence depicted in SEQ ID NO:

5.

19. The protein of claim 8 wherein Escherechia coli Fpapy-DNA glycosylase has the amino acid sequence depicted in SEQ ID NO:

23.

20. The protein of claim 8 wherein Escherechia coli endonuclease III has the amino acid sequence depicted in SEQ ID NO:

25.

21. The protein of claim 15 wherein Escherechia coli endonuclease IV has the amino acid sequence depicted in SEQ ID NO:

31.

22. A recombinant chimeric protein having the formula A-L-B or B-L-A, wherein:

A is a peptide having DNA mutation binding activity;

L is a linker peptide; and

B is a peptide having nuclease activity.

23. The chimeric protein of claim 22 wherein said linker peptide comprises from 1-30 amino acids.

24. The chimeric protein of claim 23 wherein said linker peptide comprises from 6 to 10 amino acids.

25. The chimeric protein of claim 22 wherein said DNA mutation is selected from the group consisting of single base pair mutations, point mutations, DNA mismatches, DNA insertions, DNA deletions, DNA transversions, DNA transitions, frameshift mutations and damaged DNA.

26. The chimeric protein of claim 22 wherein said DNA mutation is a deaminated base.

27. The chimeric protein of claim 22 wherein said DNA mutation is uracil incorporated DNA.

28. The chimeric protein of claim 22 wherein A is a peptide selected from the group consisting of MutS homologue2, xeroderma pigmentosum complementation group A, xeroderma pigmentosum C, xeroderma pigmentosum complementation group E, Thermus thermophilus Mut S, thymine DNA glycosylase, Escherechia coli Fpapy-DNA glycosylase, Escherechia coli endonuclease III, Escherechia coli exonuclease III, Escherechia coli endonuclease IV, T4 endonuclease, Escherechia coli uracil DNA glycosylase, Escherechia coli A/G-specific adenine DNA glycosylase, Escherechia coli Uvr A and Escherechia coli Uvr B.

29. The chimeric protein of claim 22 wherein B is a peptide selected from the group consisting of the N-terminus of human excision repair cross-complementing rodent repair deficiency, Serratia marcescens nuclease, Escherechia coli Fpapy-DNA glycosylase;

Escherechia coli endonuclease III;

Escherechia coli endonuclease IV;

T4 endonuclease;

Escherechia coli uracil DNA glycosylase;

Escherechia coli A/G-specific adenine DNA glycosylase, Esherechia coli Uvr B nuclease and Esherechia coli Uvr C nuclease.

30. An isolated and purified chimeric protein comprising a pair of proteins wherein said pair of proteins are selected from the group consisting of XPF and XPA, XPF and hMSH2, XPA and XPF, hMSH2 and XPF, Nuc and hMSH2, Nuc and XPA, MutS and XPF, XPF and MutS, Nuc and MutS, XPA-and XPF and Nuc and XPA, wherein XPF is human excision repair cross-complementing rodent repair deficiency complementation group 4 protein, XPA is xeroderma pigmentosum complementation group A protein, hMSH2 is human MutS homologue2 protein, Nuc is Serratia marcescens nuclease and MutS is Thermus thermophilus MutS.

31. The chimeric protein of claim 30 wherein said pair of proteins further includes a linker peptide.

32. The protein of claim 30 wherein XPF has the amino acid sequence depicted in SEQ ID NO:

13 or SEQ ID NO;

11.

33. The protein of claim 31 wherein the linker peptide has the amino acid sequence depicted in SEQ ID NO:

17.

34. The protein of claim 30 wherein hMSH2 has the amino acid sequence depicted in SEQ ID NO:

1or SEQ ID NO;

3.

35. The protein of claim 30 wherein Nuc has the amino acid sequence depicted in SEQ ID NO:

5.

36. The protein of claim 30 wherein MutS has the amino acid sequence depicted in SEQ ID NO:

15.

37. An isolated and purified nucleic acid encoding a chimeric polypeptide comprising a DNA mutation binding protein and a nuclease.

38. A nucleic acid construct comprising the nucleic acid of claim 37.

39. The nucleic acid construct of claim 38 wherein said nucliec acid is operably associated with an expression control sequence functional in a microbial cell.

40. The nucleic acid construct of claim 39 wherein said cell is a bacterial cell.

41. A recombinant bacterial cell comprising the nucleic acid construct of claim 38.

42. An isolated and purified nucleic acid encoding a chimeric protein having the formula A-L-B or B-L-A, wherein:

A is a peptide having DNA mutation binding activity;

L is a linker peptide; and

B is a peptide having nuclease activity.

43. The nucleic acid of claim 42 wherein A is a peptide selected from the group consisting of MutS homologue2, xeroderma pigmentosum complementation group A, xeroderma pigmentosum complementation group C, xeroderma pigmentosum complementation group E, Thermus thermophilus Mut S, thymine DNA glycosylase, Escherechia coli Fpapy-DNA glycosylase, Escherechia coli endonuclease III, Escherechia coli exonuclease III, Escherechia coli endonuclease IV, T4 endonuclease, Escherechia coli uracil DNA glycosylase, Escherechia coli A/G-specific adenine DNA glycosylase and Escherechia coli Uvr A DNA mutation binding protein and Escherechia coli Uvr B DNA mutation binding protein.

44. The nucleic acid of claim 42 wherein B is a peptide selected from the group consisting of human excision repair cross-complementing rodent repair deficiency complementation group 4, Serratia marcescens nuclease, Escherechia coli Fpapy-DNA glycosylase;

Escherechia coli endonuclease III;

Escherechia coli endonuclease IV;

T4 endonuclease;

Escherechia coli uracil DNA glycosylase Escherechia coli A/G-specific adenine DNA glycosylase, Escherechia coli Uvr B nuclease and Escherechia coli Uvr C nuclease.

45. The nucleic acid of claim 43 wherein MutS homolog 2 has the nucleotide sequence depicted in SEQ ID NO:

2 or SEQ ID NO;

4.

46. The nucleic acid of claim 43 wherein xeroderma pigmentosum complementation group A has the nucleotide sequence depicted in SEQ ID NO:

8 or SEQ ID NO;

10.

47. The nucleic acid of claim 43 wherein xeroderma pigmentosum complementation group A has the sequence depicted in SEQ ID NO:

8.

48. The nucleic acid of claim 43 wherein xeroderma pigmentosum complementation group C has the sequence SEQ ID NO:

20.

49. The nucleic acid of claim 43 wherein xeroderma pigmentosum complementation group E has the nucleotide sequence depicted in SEQ ID NO:

22.

50. The nucleic acid of claim 43 wherein Thermus thermophilus Mut S has the nucleotide sequence depicted in SEQ ID NO:

16.

51. The nucleic acid of claim 43 wherein thymine DNA glycosylase has the nucleotide sequence depicted in SEQ ID NO:

42.

52. The nucleic acid of claim 43 wherein Escherechia coli Fpapy-DNA glycosylase has the nucleotide sequence depicted in SEQ ID NO:

24.

53. The nucleic acid of claim 43 wherein Escherechia coli endonuclease III has the nucleotide sequence depicted in SEQ ID NO:

26.

54. The nucleic acid of claim 43 wherein Escherechia coli exonuclease III has the nucleotide sequence depicted in SEQ ID NO:

30.

55. The nucleic acid of claim 43 wherein Escherechia coli endonuclease IV has the nucleotide sequence depicted in SEQ ID NO:

32.

56. The nucleic acid of claim 43 wherein T4 endonuclease has the nucleotide sequence depicted in SEQ ID NO:

34.

57. The nucleic acid of claim 43 wherein Escherechia coli uracil DNA glycosylase has the nucleotide sequence depicted in SEQ ID NO:

36.

58. The nucleic acid of claim 43 wherein Escherechia coli A/G-specific adenine DNA glycosylase has the nucleotide sequence depicted in SEQ ID NO:

38.

59. The nucleic acid of claim 44 wherein Serratia marcescens nuclease has the nucleotide sequence depicted in SEQ ID NO:

6.

60. A method of detecting a DNA sequence variation in a polynucleotide, comprising:

a) obtaining said polynucleotide;

b) obtaining a chimeric protein wherein said chimeric protein has a DNA mutation binding region and nuclease region wherein said DNA mutation binding region recognizes mutated DNA;

c) forming a mixture of said polynucleotide and said chimeric protein;

d) forming a reacted sample by incubating said mixture under conditions wherein if said polynucleotide includes mutated DNA, said DNA mutation binding region binds to said mutated DNA and said nuclease cuts said mutated DNA; and

e) analyzing said reacted sample to determine the extent of cleavage of said polynucleotide to detect said DNA mutation.

61. The method of claim 60 wherein said sequence variation is a DNA mutation.

62. The method of claim 61 wherein said DNA mutation is mismatched DNA.

63. The method of claim 61 wherein said DNA mutation is uracil incorporated DNA.

64. The method of claim 61 wherein said DNA mutation is selected from the group consisting of single base pair mutations, point mutations, DNA mismatches, DNA insertions, DNA deletions, DNA transversions, DNA transitions, frameshift mutations and damaged DNA.