DP04 polymerase variants
Recombinant DP04-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 incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DP04 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 80% identical to amino acids 1-340 of SEQ ID NO:1, which recombinant DNA polymerase comprises mutations at amino acid positions 42, 56, 76, 78, 79, 82, 83, 86, 152, 155, 156, 184, 189, 248, 289, 290, 291, 292, and 293, wherein identification of positions is relative to wildtype DPO4 polymerase (SEQ ID NO:1), wherein the mutations are amino acid substitutions, wherein the mutation at position 156 is selected from the group consisting of D156R, D156L, D156A, D156G, D156S, D156I, D156M, and D156T, wherein amino acids 341-352 relative to wildtype DPO4 polymerase (SEQ ID NO:1) is deleted, and which recombinant DNA polymerase exhibits polymerase activity.
2. The recombinant DNA polymerase of claim 1 , wherein the mutation at position 42 is A42V, the mutation at position 56 is K56Y, the mutation at position 76 is M76W, the mutation at position 78 is K78N, the mutation at position 79 is E79L, the mutation at position 82 is Q82W, the mutation at position 83 is Q83G, the mutation at position 86 is S86E, the mutation at position 152 is selected from the group consisting of K152E, K152G, K152L, K152Q, K152A, K152Y, K152V, and K152M, the mutation at position 155 is selected from the group consisting of A155G, A155M, and A155C, the mutation at position 184 is P184L, the mutation at position 189 is I189W, the mutation at position 289 is V289W, the mutation at position 290 is T290K or T290R, the mutation at position 291 is E291S, the mutation at position 292 is D292Y, and the mutation at position 293 is L293W.
3. A composition comprising a recombinant DNA polymerase as set forth in claim 1 .
4. The composition of claim 3 , wherein the composition is present in a DNA sequencing system that comprises at least one non-natural nucleotide analog substrate.
5. A modified nucleic acid encoding a modified DPO4-type DNA polymerase as set forth in claim 1 .