Thermostable DNA polymerases in replication, repair and biotechnology

Abstract

Many archaea contain a unique DNA polymerase, DNA Pol D. This enzyme is a heterodimer composed of a large subunit (polymerase) and a small subunit (3’- 5’ proof reading exonuclease). The enzyme from Pyroccocus furiosus is inhibited by the presence of uracil in template strands. This research has shown that a single uracil located as far as 134 bases ahead of the primertemplate junction causes inhibition of replication. Further, using replication fork mimics, it is shown that, as expected, uracil on a template strand being copied by Pol D causes inhibition. Surprisingly, though, the presence of uracil on a complementary non-copied strand is also inhibitory. A model for uracil recognition by Pol D is proposed. The biochemical properties of the individual, large and small, subunits of the Pol D heterodimer were analysed. Both subunits were found to possess activity when expressed alone although the activity was greatly reduced compared to the Pol D heterodimer. It was not possible to regain the level of activity observed in the Pol D holoenzyme by mixing the two subunits in vitro. This finding contributed to the hypothesis that the carboxyl-terminal region of the large subunit contains an Fe-S cluster that is lost when the protein is purified aerobically. Attempts were made to express Pol D in archaeal hosts and purify the protein with the correct metallo-status; regrettably, these were not successful. Two thermostable bacterial family-B (pol II) DNA polymerases were cloned and expressed in E.coli and their biochemical properties analysed. The enzymes were found to possess many properties that make them amenable to biotechnology: polymerase activity, 3’-5’ proofreading activity, high fidelity rates and the ability to bypass uracil located in template strand DNA. Unfortunately, thermostability assays revealed that the polymerases denatured on exposure to temperatures ~85°C, making them unsuitable in the PCR. Thus, further manipulation is required to determine whether the polymerases have applications in biotechnology.