T7 DNA polymerase

T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones. The T7 DNA polymerase requires a host factor, E. coli thioredoxin, in order to carry out its function. This helps stabilize the binding of the necessary protein to the primer-template to improve processivity by more than 100-fold, which is a feature unique to this enzyme. It is a member of the Family A DNA polymerases, which include E. coli DNA polymerase I and Taq DNA polymerase. This polymerase has various applications in site-directed mutagenesis as well as a high-fidelity enzyme suitable for PCR. It has also served as the precursor to Sequenase, an engineered-enzyme optimized for DNA sequencing. Figure 2. Nucleotidyl transfer by DNA polymerase. T7 DNA polymerase catalyzes the phosphoryl transfer during DNA replication of the T7 phage. As shown in Figure 2, the 3’ hydroxyl group of a primer acts as a nucleophile and attacks the phosphodiester bond of nucleoside 5’-triphosphate (dTMP-PP). This reaction adds a nucleoside monophosphate into DNA and releases a pyrophosphate (PPi). Generally, the reaction is metal-dependent and cations such as Mg2+ are often present in the enzyme active site. For T7 DNA polymerase, the fingers, palm and thumb (Figure 1) position the primer-template so that the 3’-end of the primer strand is positioned next to the nucleotide-binding site (located at the intersection of the fingers and thumb). The base pair formed between the nucleotide and the template base fits nicely into a groove between the fingers and the 3’-end of the primer. Two Mg2+ ions form an octahedral coordinate network with oxygen ligand and also bring the reactive primer hydroxyl and the nucleotide α-phosphate close together, thereby lowering the entropic cost of nucleophilic addition. The rate-limiting step in the catalytic cycle occurs after the nucleoside triphosphate binds and before it is incorporated into the DNA (corresponding to the closure of the fingers subdomain around the DNA and nucleotide).