Enzyme derived from thermophilic organisms that functions as a chromosomal replicase, and preparation and uses thereof
A DNA Polymerase has been identified in a thermophile that functions as a chromosomal replicase. The specific enzyme is a holoenzyme III that has been identified in Thermus thermophilus, and corresponds to Polymerase III in E. coli . The genes and the polypeptides corresponding to T.th. γ, τ, ε, α and β subunits that they encode are disclosed, as are probes, vectors, methods of preparation and the methods of use. The enzymes of the present invention and their components are particularly well suited for use in procedures for the preparation of DNA, such as PCR, because of the speed and accuracy that they are able to achieve.
1. An isolated polynucleotide encoding an ε subunit of a DNA polymerase III-type enzyme of a Thermus bacterium, the encoded ε subunit possessing DNA polymerase-associated 3′-5′ exonuclease activity, wherein said isolated polynucleotide is at least 95 percent identical to the nucleotide sequence of SEQ ID NO: 94.
2. A vector comprising the polynucleotide according to claim 1 .
3. A host cell comprising the vector according to claim 2 .
4. The host cell according to claim 3 , wherein the host cell is a prokaryotic cell.
5. The isolated polynucleotide according to claim 1 , wherein the amino acid sequence encoded by the polynucleotide is at least 95 percent identical to the amino acid sequence of SEQ ID NO:95.
6. An isolated polynucleotide encoding an ε subunit of a DNA polymerase III-type enzyme of a thermophilic bacterium, wherein the polynucleotide comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 95.
7. The polynucleotide according to claim 6 , wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 94.
8. A vector comprising the polynucleotide according to claim 6 .
9. A host cell comprising the vector according to claim 8 .
10. The host cell according to claim 9 , wherein the host cell is a prokaryotic cell.
11. A method of producing a recombinant ε subunit of a DNA polymerase III-type enzyme, said method comprising:
transforming a host cell with the heterologous polynucleotide according to claim 6 under conditions suitable for expression of the ε subunit; and
isolating the ε subunit.