Prokaryotic dna repair ligases

Abstract

The present invention relates to the cloning and characterisation 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 a prokaryotic DNA repair ligase polypeptide.   
     
     
         2 . A method according to  claim 1  wherein the prokaryotic DNA repair ligase polypeptide comprises one or more of: a primase domain, a nuclease domain, and a ligase domain, said one or more domains sharing greater than 20% sequence identity with the corresponding domain sequence of Mt-Lig (CAB08492). 
     
     
         3 . A method according to  claim 1  wherein the prokaryotic DNA repair ligase polypeptide shares greater than 20% sequence identity with the sequence of Mt-Lig (CAB08492). 
     
     
         4 . A method according to  claim 1  wherein the prokaryotic DNA repair ligase polypeptide is Mt-Lig (CAB08492) or a variant or allele thereof. 
     
     
         5 . A method according to  claim 1  wherein the nucleic acid molecule and the Mt-Lig polypeptide are contacted in the presence of a prokaryotic Ku polypeptide. 
     
     
         6 . A method according to  claim 5  wherein the prokaryotic Ku polypeptide shares greater than 20% sequence identity with the sequence of Mt-Ku (CAB08491). 
     
     
         7 . A method according to  claim 6  wherein the prokaryotic Ku polypeptide is Mt-Ku (CAB08491) or an allele or variant thereof. 
     
     
         8 . A method of ligating nucleic acid molecule ends comprising;
 contacting a first nucleic acid end and a second nucleic acid end with an prokaryotic DNA repair ligase polypeptide,   wherein said first and said second nucleic acid ends are non-compatible.   
     
     
         9 . A method according to  claim 8  wherein said first and said second nucleic acid ends comprise non-complementary overhang regions. 
     
     
         10 . A method according to  claim 8  wherein the first end is on a first nucleic acid molecule and the second end is on a second nucleic acid molecule. 
     
     
         11 . A method according to  claim 10  wherein the first and second nucleic acid molecules are DNA. 
     
     
         12 . A method according to  claim 10  wherein the first nucleic acid molecule is DNA and the second nucleic acid molecule is RNA. 
     
     
         13 . A method according to  claim 8  wherein the first and second ends are on the same nucleic acid molecule. 
     
     
         14 . A method according to  claim 8  comprising isolating and/or purifying the ligated nucleic acid molecule. 
     
     
         15 . A method of labelling a nucleic acid molecule comprising;
 contacting a nucleic molecule having a first terminus with an prokaryotic DNA repair ligase polypeptide in the presence of labelled nucleotides.   
     
     
         16 . A method according to  claim 15  wherein the nucleotides are NTPs. 
     
     
         17 . A method according to  claim 15  wherein the nucleotides are dNTPs. 
     
     
         18 . 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 prokaryotic DNA repair ligase polypeptide.   
     
     
         19 . A method according to  claim 18  wherein said nucleic acid molecule and said prokaryotic DNA repair ligase polypeptide are contacted in the presence of NTPs. 
     
     
         20 . A method according to  claim 18  wherein said nucleic acid molecule and said prokaryotic DNA repair ligase polypeptide are contacted in the presence of dNTPs. 
     
     
         21 . A method of removing a single stranded overhang from the end of a nucleic acid molecule comprising;
 contacting said nucleic acid molecule with a prokaryotic DNA repair ligase polypeptide   
     
     
         22 . A method according to  claim 21  wherein the prokaryotic DNA repair ligase polypeptide is an Mt-Lig polypeptide. 
     
     
         23 . A method according to  claim 21  wherein said nucleic acid molecule is contacted in the presence of Mg 2+  or Mn 2+ . 
     
     
         24 . A method of producing an RNA molecule comprising;
 contacting a prokaryotic DNA repair ligase polypeptide and a template DNA strand in the presence of NTPs.   
     
     
         25 . A method according to  claim 24  wherein prokaryotic DNA repair ligase and template DNA are contacted in the presence of a primer oligonucleotide. 
     
     
         26 . A method of producing a DNA molecule comprising;
 contacting A prokaryotic DNA repair ligase polypeptide and a nucleic acid template in the presence of dNTPs and a primer oligonucleotide.   
     
     
         27 . A method according to  claim 26  wherein the nucleic acid template is an RNA template. 
     
     
         28 . A method according to  claim 26  wherein the nucleic acid template is an DNA template. 
     
     
         29 . A method according to  claim 8  wherein the prokaryotic DNA repair ligase polypeptide comprises one or more of: a primase domain, a nuclease domain, and a ligase domain, said one or more domains sharing greater than 20% sequence identity with the corresponding domain sequence of Mt-Lig (CAB08492). 
     
     
         30 . A method according to  claim 8  wherein the prokaryotic DNA repair ligase polypeptide shares greater than 20% sequence identity with the sequence of Mt-Lig (CAB08492). 
     
     
         31 . A method according to  claim 8  wherein the prokaryotic DNA repair ligase polypeptide is Mt-Lig (CAB08492) or a variant or allele thereof. 
     
     
         32 . A method according to  claim 8  wherein the nucleic acid molecule and the Mt-Lig polypeptide are contacted in the presence of a prokaryotic Ku polypeptide. 
     
     
         33 . A method according to  claim 32  wherein the prokaryotic Ku polypeptide shares greater than 20% sequence identity with the sequence of Mt-Ku (CAB08491). 
     
     
         34 . A method according to  claim 32  wherein the prokaryotic Ku polypeptide is Mt-Ku (CAB08491) or an allele or variant thereof. 
     
     
         35 . A kit comprising an isolated Mt-Lig polypeptide for use in a method according to  claim 1 . 
     
     
         36 . A kit according to  claim 35  comprising an isolated Mt-Ku polypeptide. 
     
     
         37 . A kit according to  claim 35  comprising dNTPs. 
     
     
         38 . A kit according to  claim 35  comprising NTPs. 
     
     
         39 . A kit according to  claim 35  comprising one or more of buffers, stabilisers and excipients. 
     
     
         40 . A method of producing a prokaryotic DNA repair polypeptide comprising;
 (a) causing expression from nucleic acid which encodes a prokaryotic DNA repair polypeptide in a suitable expression system to produce the polypeptide recombinantly; and, testing the recombinantly produced polypeptide for prokaryotic DNA repair activity.   
     
     
         41 . A method according to  claim 40  wherein the recombinantly produced polypeptide is tested for one or more of: non-complementary end ligation activity, DNA dependent RNA primase activity, 3′-5′ exonuclease activity, DNA and RNA dependent DNA polymerase activity, DNA dependent RNA polymerase activity, ATP dependent DNA and RNA ligase activity and DNA terminal transferase activity. 
     
     
         42 . A method according to  claim 39  wherein the prokaryotic DNA repair polypeptide is an Mt-Lig polypeptide or an allele or variant thereof. 
     
     
         43 . A method according to  claim 40  comprising purifying said recombinantly produced polypeptide.