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

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 impro ving ddNTP utilisation, 'Rational' alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural inf ormation) did not yield the desired level of selectivity for ddNTPs. Howeve r, alteration at four positions (Q472, A486, L490 and Y497) gave rise to va riants which incorporated ddNTPs better than the wild type, allowing sequen cing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild-type Pf u-Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490 W), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y rep resenting a 150-fold improvement over the wild type, A486, L490 and Y497 ar e 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 th at 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 i n conformation that subtly alter the nucleotide triphosphate binding site s uch that ddNTPs become favoured; (ii) interference with a conformational ch ange that may be critical both for the polymerisation step and discriminati on between different nucleotide triphosphates.