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. 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). The amino acids present in the active site assist in creating a stabilizing environment for the reaction to proceed. Amino acids such as Lys522, Tyr526, His506 and Arg518 act as hydrogen bond donors. The backbone carbonyl of Ala476, Asp475 and Asp654 form coordinate bonds with the Mg2+ ions. Asp475 and Asp654 form a bridge with the Mg2+ cations to orient them properly. The Mg2+ ion on the right (Figure 3) interacts with negatively charged oxygens of the alpha(α), beta(β) and gamma(γ) phosphates to align the scissile bond for the primer to attack. Even if there is no general base within the active site to deprotonate the primer hydroxyl, the lowered pka of the metal-bound hydroxyl favors the formation of the 3’-hydroxide nucleophile. Metal ions and Lys522 contact non-bridging oxygens on the α-phosphate to stabilize the negative charge developing on the α-phosphorus during bond formation with the nucleophile. Moreover, the Lys522 sidechain also moves to neutralize the negatively charged pyrophosphate group. Tyr526, His506, Arg518 side chains and the oxygen from the backbone carbonyl group of Ala476 take part in the hydrogen bond network and assist in aligning the substrate for phosphoryl transfer.