Novel DNA glycosylases and their use

• US 20060134631A1
• Filed: 12/23/2003
• Published: 06/22/2006
• Est. Priority Date: 01/09/1996
• Status: Active Grant

• Current Assignee: Bodil Kavli, Clifford D. Mol, Geir Slupphaug, Hans E. Krokan, John A. Tainer
• Original Assignee: Bodil Kavli, Clifford D. Mol, Geir Slupphaug, Hans E. Krokan, John A. Tainer
• Inventors: Tainer, John A., Krokan, Hans E., Mol, Clifford D., Slupphaug, Geir, Kavli, Bodil
• Granted Patent: US 7,662,601 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/2497 hydrolysing N- glycosyl compounds (3.2.2)
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Y 302/02027 Uracil-DNA glycosylase (3.2.2.27)

Abstract

Novel cytosine-, thymine- and uracil-DNA glycosylases, subcellular localization peptides, nucleic acid molecules containing the same, methods of identifying such enzymes and their use in various methods including mutagenesis, cell killing and DNA sequencing and modification, are desired.

Claims

1. An isolated thymine-DNA glycosylase (TDG) capable of releasing thymine bases from single stranded (ss) DNA and double stranded (ds) DNA,

said TDG obtainable by modification of a uracil-DNA glycosylase (UDG), wherein tyrosine (Tyr) at amino acid position 147 is substituted or modified in the human UDG protein encoded

a) by the human uracil nucleic acid glycosylase gene (UNG1) (SEQ ID NO:

8), or

b) by the alternatively spliced human uracil nucleic acid glycosylase gene (UNG2) (SEQ ID NO;

     2), or

wherein an equivalent residue in a homologous UDG of another species is substituted or modified.

2. A TDG as claimed in claim 1 wherein said Tyr or equivalent residue is replaced with an alanine residue (Ala).

3. A thymidine-DNA glycosylase (TDG) as claimed in claim 1 wherein said TDG is capable of releasing both thymidine and uracil bases from ssDNA and dsDNA.

4. A TDG as claimed in claim 1 wherein said TDG is capable of releasing thymine bases from A:

T DNA pairs.

5. A TDG as claimed in claim 1 wherein said UDG which is modified is human.

6. A DNA glycosylase as claimed in claim 1 which additionally comprises

at least one nuclear localization peptide sequence encoded by a nucleic acid molecule comprising the sequence

(SEQ. ID. No.:

7. A DNA glycosylase as claimed in claim 6 wherein said nuclear localizing peptide includes the amino acid sequence wherein RKR is followed by histidine.

8. A method of performing enzymatic DNA sequencing to determine the position of thymidine bases comprising the step of treating said DNA with at least one TDG as defined in claim 1.

9. A method of performing in vitro mutagenesis which method comprises introducing one or more TDG of claim 1 to a complex sample comprising a nucleic acid substrate, wherein thymidine is removed and optionally replaced in said nucleic acid substrate.

10. A method of performing in vivo mutagenesis which method comprises introducing a TDG of claim 1 into a liposome, and introducing said liposome into a suitable host cell.

11. A method of removing contaminating DNA from a sample prior to PCR amplification, comprising introducing one or more TDG of claim 1 to a reaction mixture, wherein contaminating DNA are digested by said one or more TDG, and

inactivating said one or more TDG prior to addition of a DNA sample and amplification.

12. A method of producing randomly fragmented DNA comprising introducing one or more TDG of claim 1 to a DNA sample to yield one or more apyrimidinic site(s) (AP-site(s)) in said sample, and

treating said sample with an alkaline solution alone, an apurinic/apyrimidinic-site endonuclease (AP-endonuclease), or a combination thereof, wherein breaks are produced in the DNA at the AP-site(s) which yield randomly fragmented DNA of defined size ranges.

13. An assay for the identification of the TDG of claim 1 in a sample, which comprises determining any activity in the sample of excising thymine and optionally also uracil from an introduced ssDNA and/or dsDNA substrate.

14. A nucleic acid molecule comprising a nucleotide sequence which encodes a TDG of claim 1 or which comprises the complement of said nucleic acid sequence.

15. An expression vector containing the nucleic acid molecule of claim 14.

16. A transformed or transfected host cell comprising the nucleic acid molecule of claim 15.

17. A method to kill cells, comprising introducing a nucleic acid molecule encoding the TDG of claim 1 into a cell and expressing said TDG in the cell to a level that results in said cell being killed.

18. A method to modify DNA which method comprises contacting said DNA with the TDG of claim 1.