Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-11-17
2001-12-25
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S320100, C435S252300, C435S325000, C435S091100, C435S091200, C530S350000, C536S023200
Reexamination Certificate
active
06333183
ABSTRACT:
FIELD OF INVENTION
The instant disclosure pertains to DNA polymerase mutants from Pyrococcus furiosus which exhibit improved dideoxynucleotide utilization.
BACKGROUND OF INVENTION
A polymerases constitute a core component of DNA sequencing methods, a widespread and important biotechnology, based on chain-termination by dideoxynucleotide-triphosphates (ddNTPs), either the ddNTPs themselves or fluorescent derivatives. Discrimination between chain-terminating ddNTPs and dNTPs plays a key role in DNA sequencing performance. Effective ddNTP incorporation is associated with a high uniformity of signal intensity in sequencing ladders. Furthermore, efficient usage of ddNTPs requires lower concentrations; an advantage when fluorescent terminators are used, as large excesses give rise to high backgrounds. Bacteriophage T7 DNA polymerase incorporates ddNTPs much more efficiently than the enzymes from
E. coli
and
T. aquaticus
and, as a consequence, gives superior sequencing ladders. The molecular basis for discrimination between dNTPs and ddNTPs resides in a single amino acid, at an equivalent location: Y526 (T7); F762 (
E. coli
); F667 (
T. aquaticus
). The T7 mutant, Y526F, shows a much reduced ability to use ddNTPs and, consequently, gives poor sequencing ladders. The F762Y and F667Y variants of
E. Coli
and
T. aquaticus
use ddNTPs effectively and show much improved sequencing properties. The important role of F762, in the
E. coli
polymerase, for deoxynucleotide-triphosphate recognition has been confirmed by a more complete kinetic analysis. Mutants of
T. aqualicus
DNA polymerase which have exonuclease activity removed, and contain tyrosine at position 667, have excellent sequencing properties and are perhaps the most widely used enzymes for DNA sequencing; these include Thermo-Sequenase™ and Amplitaq FS™ DNA polymerases. All three polymerases belong to the Pol-I family (also called family A polymerases) and the critical aromatic amino acid is found in a highly conserved stretch, the B-motif (also called region III)(FIG.
1
). Structural data has shown that the B-motiff amino acids form an &agr;-helix (the O-helix in the case of
E. coli
) with the most conserved amino acids on one side of the helix, forming part of the dNTP binding site (FIG.
1
). The tyrosine/phenylalanine is near the sugar of the dNTP, rationalising its critical role in dNTP/ddNTP selection.
Polymerases with thermal stability are routinely used for DNA sequencing. Not only are they generally more robust than mesophilic enzymes but are essential for cycle-sequencing protocols, which involve heat-cool cycles. The extreme thermostability of polymerases purified from hyper-thermophilic archaea suggest these enzymes have potential use in DNA sequencing. However, archaeal polymerases often use ddNTPs poorly and, as a result, are generally not as useful in DNA sequencing as, for example, Thermo Sequenase™ DNA polymerase. Archaeal polymerascs belong to the &agr;-family (also called the B-family, or Pol-II family), a different group to the better characterised Pol-I enzymes. However, sequence alignment shows that the Pol-&agr; family also has a B-motif, even though it cannot be exactly aligned with that of Pol-I (FIG.
1
), and there is no exact counterpart to the aromatic amino acid critical for ddNTP/dNTP selection in the Pol-I family. Nevertheless, mutations in the B-motif of &agr;-polymerases influence dNTP binding, suggesting a role in deoxynucleoside-triphosphate recognition. Recently, crystal structures of two Pol-&agr; members, bacteriophage RB69 gp43 and
Thermococcus gorgonarius
(Tgo), have been published. The B-motif amino acids form an &agr;-helix (the P-helix with both enzymes) which, as suggested by sequence alignments, is similar, but not structurally identical to the corresponding &agr;-helix in the Pol-I family (FIG.
1
). Although both structures lack bound nucleic acid it was possible to model primer-template and dNTP into RB69. The B-region was located near both primer/template and dNTP but did not appear to provide an amino acid that binds near the sugar of the dNTP. Rather, this might be supplied by tyrosine 416, an amino acid from another part of the polymerase, which packs under the sugar ring of the dNTP.
Sequence and structural comparisons of Pol-I and Pol-&agr; members indicate that homologous regions are used for dNTP binding and recognition. However, the exact details of the interaction with dNTPs, and hence discrimination between dNTPs and ddNTPs, differs between the two classes. Therefore, the simple tyrosine/phenylalanine switch, so successful in converting
T. aquaticus
to a good sequencing polymerase, is unlikely to be possible with archaeal polymerases.
SUMMARY OF INVENTION
The instant invention comprises
Pyrococcus furiosus
polymerase mutants that recognise ddNTPS a factor of up to 150 fold better and are superior for cycle-sequencing protocols than the wild type enzyme. Methods for the isolation and characterisation of these mutants are discussed. The mutants exhibit superior thermal stability compared to other thermostable DNA polymerases, exhibiting stability for hours at 95° C. (compared with 30-45 minutes for
t. aquaticus
polymerase), thereby permitting a greater number of cycles at elevated temperature and, hence, enhanced sensitivity.
REFERENCES:
patent: 5489523 (1996-02-01), Mathur
patent: 5827716 (1998-10-01), Mamone
patent: 0 655 506 A1 (1995-05-01), None
patent: 0 727 496 A2 (1996-08-01), None
patent: WO 99/06538 (1999-02-01), None
Evans, S., et al. “Improving Dideoxynucleotide-Triphosphate Utilisation by the Hyper-Thermophilic DNA Polymerase from the ArchaeonPyrococcus furiosus” Nucleic Acids Research, vol. 28, No. 5, Mar. 1, 2000, pp. 1059-1066.
Gardner, A., et al. “Determinants of Nucleotide Sugar Recognition in an Archaeon DNA Polymerase” Nucleic Acids Research, vol. 27, No. 12, Jun. 15, 1999, pp. 2545-2553.
Connolly Bernard A.
Davis Maria
Evans Steven
Mamone Joseph Anthony
Amersham Pharmacia Biotech Inc.
Horlick Kenneth R.
Ronning, Jr. Royal N.
Spiegler Alexander H.
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