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Nucleic Acids Research, 2000, Vol. 28, No. 5 1059-1066
© 2000 Oxford University Press

Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus

Steven J. Evans, Mark J. Fogg, Anthony Mamone1, Maria Davis1, Laurence H. Pearl2 and Bernard A. Connolly*

Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne NE2 4HH, UK, 1Amersham Pharmacia Biotech Inc., 800 Centennial Avenue, Piscataway, NJ 08855, USA and 2Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK

Polymerases from the Pol-I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol-II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. ‘Rational’ alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild-type Pfu–Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490W), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type. A486, L490 and Y497 are on an {alpha}-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this {alpha}-helix to a second long helix. None of the four amino acids can contact the dNTP directly. Therefore, the increased selectivity for ddNTPs is likely to arise from two factors: (i) small overall changes in conformation that subtly alter the nucleotide triphosphate binding site such that ddNTPs become favoured; (ii) interference with a conformational change that may be critical both for the polymerisation step and discrimination between different nucleotide triphosphates.

* To whom correspondence should be addressed. Tel: +44 191 222 7371; Fax: +44 191 222 7424; Email: b.a.connolly@ncl.ac.uk Present address: Laurence H. Pearl, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK


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