DNA glycosylases and their use

• US 7,662,601 B2
• Filed: 12/23/2003
• Issued: 02/16/2010
• Est. Priority Date: 01/09/1996
• Status: Expired due to Fees

• 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
• Primary Examiner(s): Pak; Yong D
• Application Number:
  US10/746,239
• Publication Number:
  US 20060134631A1
• Time in Patent Office: 2,247 Days
• Field of Search: None
• US Class Current: 435/193
• 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) which releases thymine bases from single stranded (ss) DNA and double stranded (ds) DNA, said TDG obtainable by modification of a uracil-DNA glycosylase (UDG), wherein said modification consists of replacing tyrosine (Tyr) at amino acid position 147 with a different amino acid in the human UDG protein which protein comprises SEQ ID NO:

8.

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

3. The thymine-DNA glycosylase (TDG) as claimed in claim 1 wherein said TDG releases both thymine and uracil bases from ssDNA and dsDNA.

4. The TDG as claimed in claim 1 wherein said TDG releases thymine bases from A:

T DNA pairs.

5. The 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

6. The DNA glycosylase as claimed in claim 5 wherein said nuclear localizing peptide includes the amino acid sequence wherein RKR is followed by histidine.

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

8. 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 thymine is removed and optionally replaced in said nucleic acid substrate.

9. 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.

10. 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.

11. 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.

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