Two Component DNA Replicases with Modified Beta-Subunit Binding Motifs, and Uses Thereof
The invention provides Pol III α mutants, and modified Pol III replicases comprising the same, and methods of using modified Pol III replicases for a variety of nucleic acid replication reactions.
1 . A Pol III α mutant having at least one mutation in one or more of motifs A and B, wherein said modified Pol III α has altered activity relative to an unmodified Pol III α not having said at least one mutation.
2 . The Pol III α mutant according to claim 1 , wherein said Pol III α mutant has altered dNTP discrimination activity relative to said unmodified Pol III α.
3 . The Pol III α mutant according to claim 2 , wherein said Pol III α mutant has increased affinity for a ddNTP.
4 . The Pol III α mutant according to claim 2 , wherein said Pol III α mutant comprises one or more mutations in motif B.
5 . The Pol III α mutant according to claim 2 , wherein said Pol III α mutant has increased affinity for a labeled nucleotide.
6 . The Pol III α mutant according to claim 5 , wherein said Pol III α mutant comprises one or more mutations in motif A.
7 . The Pol III α mutant according to claim 5 , wherein said Pol III α mutant comprises one or more mutations in motif B.
8 . The Pol III α mutant according to claim 5 , wherein said Pol III α mutant comprises one or more mutations in motif A and one or more mutations in motif B.
9 . The Pol III α mutant according to claim 1 , wherein said Pol III α mutant has altered primer discrimination activity relative to said unmodified Pol III α.
10 . The Pol III α mutant according to claim 9 , wherein said Pol III α mutant has increased affinity for RNA-primed template.
11 . The Pol III α mutant according to claim 9 , wherein said Pol III α mutant has decreased affinity for DNA-primed template.
12 . The Pol III α mutant according to claim 9 , wherein said Pol III α mutant has increased affinity for DNA-primed template.
13 . The Pol III α mutant according to claim 9 , wherein said Pol III α mutant has decreased affinity for RNA-primed template.
14 . The Pol III α mutant according to claim 9 , wherein said Pol III α mutant comprises one or more mutations in motif B.
15 . A modified Pol III replicase, comprising a Pol III α mutant according to any one of claims 1 - 14 .
16 . The modified Pol III replicase according to claim 15 , wherein said modified Pol III replicase lacks a clamp loader.
17 . The modified Pol III replicase according to claim 15 , wherein said modified Pol III replicase comprises a β sliding clamp.
18 . A method for classifying a candidate polypeptide as a bacterial DNA Pol III α, comprising identifying in said candidate polypeptide at least one of bacterial DNA Pol III α motifs A, B or C.
19 . The method according to claim 18 , comprising identifying in said candidate polypeptide said bacterial DNA Pol III α motifs A and B.
20 . The method according to claim 19 , further comprising determining the arrangement of said bacterial DNA Pol III α motifs A and B.
21 . The method according to claim 20 , further comprising determining the spacing of said bacterial Pol III α motifs A and B.
22 . The method according to claim 19 , further comprising identifying in said candidate polypeptide bacterial DNA Pol III α motif C.
23 . The method according to claim 22 , further comprising determining the arrangement of said bacterial DNA Pol III α motifs C and A, C and B, or C and A and B.
24 . The method according to claim 23 , further comprising determining the spacing of said bacterial Pol III α motifs C and A, C and B, or C and A and B.
25 . The method according to claim 18 , wherein said bacterial DNA Pol III α motifs correspond to gram positive bacteria.
26 . The method according to claim 18 , wherein said candidate polypeptide is derived from a human sample.
27 . A nucleic acid amplification kit, comprising a Pol III α mutant according to claim 1 .
28 . A nucleic acid amplification reaction tube, comprising a Pol III α mutant according to claim 1 .
29 . A nucleic acid amplification reaction mixture, comprising a Pol III α mutant according to claim 1 .
30 . A method of replication nucleic acid, comprising subjecting said nucleic acid to a replication reaction in a replication reaction mixture comprising a Pol III α mutant according to claim 1 .
31 . A method for amplifying and sequencing nucleic acid in a single reaction mixture, comprising subjecting said nucleic acid to a simultaneous amplification and sequencing reaction in a reaction mixture comprising a Pol III α mutant according to claim 1 .
32 . A method for diagnosing a patient as having a bacterial infection, comprising identifying in a candidate polypeptide obtained from said patient bacterial DNA Pol III α motifs A, B, or C, or a combination thereof.
33 . The method according to claim 32 , comprising identifying in said candidate polypeptide at least two of said bacterial DNA Pol III α motifs.
34 . The method according to claim 33 , further comprising determining the arrangement of said at least two bacterial DNA Pol III α motifs in said candidate polypeptide.
35 . The method according to claim 34 , further comprising determining the spacing of said at least two bacterial DNA Pol III α motifs in said candidate polypeptide.
36 . The method according to claim 35 , wherein said bacterial DNA Pol III α motifs are gram positive bacterial Pol III consensus motifs.
37 . A method for diagnosing a patient as having a bacterial infection, comprising obtaining a sample from said patient, and identifying in said sample a nucleic acid comprising one or more nucleotide sequences encoding one or more of bacterial DNA Pol III α motifs A, B, and C.
38 . The method according to claim 37 , wherein said nucleic acid comprises at least two of said nucleotide sequences encoding bacterial DNA Pol III α motifs A, B, and C.