Prokaryotic DNA repair ligases
View Patent ↗The present invention relates to the cloning and characterization of a prokaryotic DNA repair ligase, which is shown to possess a range of activities that allow the ligation and repair of non-compatible DNA ends and double strand breaks (DSBs). The enzyme has a range of applications in the manipulation and cloning of nucleic acids.
1. A method of modifying a nucleic acid molecule comprising:
contacting the nucleic acid molecule with an isolated prokaryotic DNA ligase polypeptide in the presence of a prokaryotic Ku polypeptide, wherein the prokaryotic DNA ligase polypeptide has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 91 and the prokaryotic Ku polypeptide has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 92.
2. A method of ligating nucleic acid molecule ends comprising:
contacting a first nucleic acid end and a second nucleic acid end with an isolated prokaryotic DNA ligase polypeptide in the presence of a prokaryotic Ku polypeptide,
wherein said first and said second nucleic acid ends comprise non-complementary overhang regions, and
wherein the prokaryotic DNA ligase polypeptide has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 91 and the prokaryotic Ku polypeptide has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 92.
3. The method according to claim 2 wherein the first end is on a first nucleic acid molecule and the second end is on a second nucleic acid molecule.
4. The method according to claim 3 wherein the first and second nucleic acid molecules are DNA.
5. The method according to claim 3 wherein the first nucleic acid molecule is DNA and the second nucleic acid molecule is RNA.
6. The method according to claim 2 wherein the first and second ends are on the same nucleic acid molecule.
7. The method according to claim 2 further comprising isolating the ligated nucleic acid molecule, purifying the ligated nucleic acid molecule, or both isolating and purifying the ligated nucleic acid molecule.
8. A method of filling in a single stranded gap in a double stranded nucleic acid molecule comprising:
contacting a double stranded nucleic acid molecule having a single stranded region with an isolated prokaryotic DNA ligase polypeptide in the presence of a prokaryotic Ku polypeptide, wherein the prokaryotic DNA ligase polypeptide has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 91 and the prokaryotic Ku polypeptide has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 92.
9. The method according to claim 8 wherein said nucleic acid molecule and said prokaryotic DNA repair ligase polypeptide are contacted in the presence of NTPs.
10. The method according to claim 8 wherein said nucleic acid molecule and said prokaryotic DNA repair ligase polypeptide are contacted in the presence of dNTPs.
11. A method of removing a single stranded overhang from the end of a nucleic acid molecule comprising:
contacting said nucleic acid molecule with an isolated prokaryotic DNA ligase polypeptide in the presence of a prokaryotic Ku polypeptide, wherein the prokaryotic DNA ligase polypeptide has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 91 and the prokaryotic Ku polypeptide has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 92.
12. The method according to claim 11 wherein said nucleic acid molecule is contacted in the presence of Mg 2+ or Mn 2+ .