7 - Modified T7 DNA Polymerase for DNA Sequencing

Abstract

When bacteriophage T7 infects its Escherichia coli host, it directs the production of a phage-specific DNA polymerase. This enzyme is a two-subunit protein. The smaller subunit, thioredoxin, is the product of the host trxA gene. The larger subunit is the product of the T7 gene 5. This enzyme has two known catalytic activities, DNA-dependent DNA polymerase and a 3′ → 5′-exonuclease activity that is active on both single-stranded and double-stranded DNA. Both of these activities are also present in purified gene 5 protein, although the DNA polymerase activity and the double-stranded exo-nuclease are both greatly increased by the binding of thioredoxin. When assayed on a single-stranded substrate, the exonuclease activity of T7 DNA polymerase hydrolyzes DNA about as fast as the polymerase can synthesize DNA under optimal polymerization conditions. T7 DNA polymerase catalyzes the incorporation of dideoxynucleotides into DNA. This DNA polymerase also catalyzes the removal of the dideoxynucleotides, resulting in little useful sequence information.