Nucleic acid molecules encoding mismatch endonucleases and methods of use thereof

Abstract

We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3′ to 5′ exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. Also described are mismatch endonucleases suitable for use in the process.

1 . An isolated protein having mismatch endonuclease activity comprising an amino acid sequence that is at least 60% identical to SEQ ID NO:17 as determined by BLAST analysis. 
     
     
         2 . The protein of  claim 1  wherein the amino acid sequence is at least 65% identical to SEQ ID NO:17 as determined by BLAST analysis. 
     
     
         3 . The protein of  claim 1  wherein the amino acid sequence is at least 70% identical to SEQ ID NO:17 as determined by BLAST analysis. 
     
     
         4 . The protein of  claim 1  wherein the amino acid sequence is at least 80% identical to SEQ ID NO:17 as determined by BLAST analysis. 
     
     
         5 . The protein of  claim 1  wherein the amino acid sequence is at least 90% identical to SEQ ID NO:17 as determined by BLAST analysis. 
     
     
         6 . The protein of  claim 1  wherein the amino acid sequence is at least 95% identical to SEQ ID NO:17 as determined by BLAST analysis.