Nucleic acid encoding bacillus stearothermophilus delta polymerase subunit

Abstract

The present invention relates to an isolated DNA molecule from a thermophilic bacterium which encodes a DNA polymerase III-type enzyme subunit. Also encompassed by the present invention are host cells and expression system including the heterologous DNA molecule of the present invention, as well as isolated replication enzyme subunits encoded by such DNA molecules. Also disclosed is a method of producing a recombinant thermostable DNA polymerase III-type enzyme, or subunit thereof, from a thermophilic bacterium, which is carried out by transforming a host cell with at least one heterologous DNA molecule of the present invention under conditions suitable for expression of the DNA polymerase III-type enzyme, or subunit thereof, and then isolating the DNA polymerase III-type enzyme, or subunit thereof.

Description

[0001] The present application is a continuation of U.S. patent application Ser. No. 09 / 716,964, filed Nov. 21, 2000, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 642,218, filed Aug. 18, 2000, as a continuation of U.S. patent application Ser. No. 09 / 057,416 filed Apr. 8, 1998, which claims the benefit of U.S. Provisional Patent Application Serial No. 60 / 043,202 filed Apr. 8, 1997, all of which are hereby incorporated by reference in their entirety.[0003] The present invention relates to thermostable DNA polymerases and, more particularly, to such polymerases as can serve as chromosomal replicases and are derived from thermophilic bacteria. More particularly, the invention extends to DNA polymerase III-type enzymes from thermophilic bacteria, including Aquifex aeolicus, Thermus thermophilus, Thermotoga maritima, and Bacillus stearothermophilus, as well as purified, recombinant or non-recombinant subunits thereof and their use, and to isolated DNA coding for ...

AI Technical Summary

Benefits of technology

[0287] As discussed above, the present invention has as one of its characterizing features, that a Polymerase III-type enzyme as defined hereinabove, has been discovered in a thermophile, that has the structure and function of a chromosomal replicase. This structure and function confers significant benefit when the enzyme is employed in procedures such as PCR where speed and accuracy of DNA reconstruction is crucial.

[0296] The discovery of a dnaX gene in T.th. provided confidence that thermophilic bacteria would contain a three component Pol III-type enzyme. Hence, we proceeded to identify the dnaQ and dnaN genes encoding, respectively, the proofreading 3'-5' exonuclease, and the .beta. DNA sliding clamp subunits of a Pol III-type enzyme. Following this, we purified from extracts of T.th. cells, a Pol III-type enzyme. This enzyme preparation had the unique property of extending a single primer around a long 7.2 kb single strand DNA genome of M13mp 18 bacteriophage. Such a primer extension assay serves as a tool to detect and identify the Pol III-type of enzyme in cell extracts. The enzyme was confirmed to be a Pol III-type enzyme based on its reactivity with antibody directed against the E. coli .alpha. subunit (the DNA polymerase subunit) and antibody directed against E. coli .gamma. subunit. Proteins corresponding to .alpha., .tau., .gamma., .delta. and .delta.' were easily visible and lend themselves to identification of the genes through use of peptide microsequencing followed by primer design for PCR amplification. For example, from this-DNA pol III-type preparation, the peptide sequence of the .alpha. subunit was obtained, which then allowed the dnaE gene encoding the .alpha. subunit (DNA polymerase) of the Pol III-type enzyme to be obtain.

[0310] The A. aeolicus .alpha..tau..delta..delta.' / .beta. Pol III can be applied in several useful DNA handling techniques. For example, the thermophilic Pol III will be useful in DNA sequencing, especially at high temperature. Also, use of a thermal resistant rapid and processive Pol III is an important improvement to polymerase chain reaction technology. The ability of the A. aeolicus Pol III to extend primers for multiple kilobases makes possible the amplification of very long segments of DNA (long chain PCR).

Problems solved by technology

Each of the references states that a drawback of the extant polymerases are their limited thermostability, and consequent useful life in the participation in PCR.

Such limitations also manifest themselves in the inability to obtain extended lengths of nucleotides, and in the instance of Taq polymerase, the lack of 3' to 5' exonuclease activity, and the drawback of the inability to excise misinserted nuicleotides,(Perrino, 1990).

However, previous work did not fully reconstitute the working replication machinery from fully recombinant subunits.

Mismatch incorporation during the synthesis or extension of the newly synthesized DNA molecule may result in one or a number of mismatched base pairs.

However, a thermostable DNA pol III-type complex used in these methods need only be added once at the start of the amplification (as for Taq DNA polymerase in traditional PCR amplifications), as its activity will be unaffected by the high temperature of the denaturation step.

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