DPO4 polymerase variants with improved accuracy
Recombinant DPO4-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and accurate incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.
1. An isolated recombinant DNA polymerase, which recombinant DNA polymerase comprises an amino acid sequence that is at least 90% identical to amino acids 1-340 of SEQ ID NO:1, which recombinant polymerase comprises a mutation at amino acid positions 63, 76, 78, 79, 82, 83, 86, 254, and 289, wherein the mutation at amino acid position 63 is E63R, the mutation at amino acid position 76 is M76W, the mutation at amino acid position 78 is K78D, the mutation at amino acid position 79 is E79L, the mutation at amino acid position 82 is Q82W, the mutation at amino acid position 86 is S86E, the mutation at amino acid position 254 is N254K or N254D, and the mutation at amino acid position 289 is V289W wherein identification of positions is relative to wildtype DPO4 polymerase (SEQ ID NO:1), and which recombinant DNA polymerase exhibits polymerase activity.
2. The polymerases of claim 1 , further comprising at least one mutation at an amino acid position selected from the group consisting of 42, 56, 152, 153, 155, 156, 184, 187, 188, 189, 190, 248, 290, 291, 292, 293, 294, 295, 296, 297, 299, 300, 301, 317, 321, 324, 325, and 327.
3. The polymerase of claim 2 , wherein the mutations at amino acid positions 42, 56, 152, 153, 155, 156, 184, 187, 188, 189, 190, 248, 290, 291, 292, 293, 294, 295, 296, 297, 299, 300, 301, 317, 321, 324, 325, and 327 are A42V, K56Y, K152L or K152A, I153T or I153V, A155G, D156R, P184L or P184Q, G187P, N188Y, I189W or I189F, T190Y, I248T, T290K, E291S, D292Y, L293W, D294N, I295S, V296Q, S297Y, G299W, R300S, T301W, K317Q, K321Q, E324K, E325K, and E327K.
4. A composition comprising a recombinant DNA polymerase as set forth in claim 1 .
5. The composition of claim 4 , wherein the composition is present in a DNA sequencing system that comprises at least one non-natural nucleotide analog substrate.
6. The polymerase of claim 1 , wherein amino acids 341-352 relative to wildtype DPO4 polymerase (SEQ ID NO: 1) are deleted.