Introduction:

Bacteriophage T7 is a lytic phage that uses E. coli as a host to replicate its DNA. Its linear chromosome is replicated by a DNA replication complex working in concert with helicase, primase, and single strand binding proteins. The replication complex is a member of the PolI family of polymerases. It contains two active sites: a 5'-3' polymerase site for addition of deoxyribonucleotides and a 3'-5' exonuclease site for proofreading. The T7 polymerase does not exhibit 5'-3' exonuclease activity.

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Enzyme Structure and DNA Binding:
The general shape of the T7 replication complex and other members of the PolI family is described as a right hand, with regions corresponding to fingers , a palm, and a thumb . The exonuclease site protrudes from one side of the hand.

The crystal structure of the T7 replication complex was determined in the presence of DNA consisting of a primer strand and a template strand . The structure also shows the location of the next dNTP to be added to the DNA. In this case, the final nucleotide on the primer strand and the incoming nucleotide are 2',3'dideoxynucleotides. The lack of a 3' -OH keeps the next polymerase reaction from occurring so the nucleotides are frozen in the polymerase active site, making structure determination of the complex possible.

Most of the DNA is in the B-form, with the exception of the final two base pairs on the 3' end of the primer. These nucleotides are in the A-form. The nucleotides at the 5'end of the template strand are turned away from the active site. The base preceding the template base stacks against His607. Binding to the enzyme bends the DNA so the primer-template can sit in the palm of the enzyme in a position which aligns the free 3' end of the primer in the polymerase active site. The incoming nucleotide is shown in ball-and-stick.

Processivity and Thioredoxin:
On its own, the DNA polymerase will incorporate less than 20 nucleotides before detaching from the primer-template. To increase its processivity, the polymerase binds to the E. coli protein thioredoxin. Binding occurs at the thumb and processivity is thought to increase due to the ability of thioredoxin to form a clamp over the DNA. Processivity is increased to such a degree that the entire T7 genome is often replicated without the dissociation and rebinding of the polymerase to the chromosome.

Specificity for Deoxynucleotides:
The polymerase's specificity for deoxynucleotides arises from the existence of a hydrophobic pocket created by the Tyr526, Glu480, and Tyr530 residues. Unfavorable electrostatic interactions prevent the binding of nucleotides with a 2' -OH. C2' is highlighted in green. In addition to the contacts shown, the beta-phosphate is also in a position to form a hydrogen bond with the a 3' -OH of the incoming nucleotide. One or both of the beta-phosphate contacts are needed for efficient polymerization.

Polymerase Activity:
Polymerization occurs by nucleophilic attack of the 3' -OH of the primer strand on the alpha-phosphate of the incoming dNTP. This occurs by a two metal mechanism that is characteristic of both prokaryotic and eukaryotic polymerases.

In T7 DNA polymerase, two Mg2+ coordinate with Asp654, Asp475, Ala476, two water molecules, and the 3' -OH and 5' phosphate groups. Hydrogen bonds are formed with the side chain carboxylates of Asp654 and Asp476 and with the backbone carbonyl or Ala476. Ser477 is shown for reference.

Metal A stabilizes the deprotonation of the 3' hydroxyl, which creates a better nucleophile. The resulting oxyanion attacks the alpha-phosphate of the dNTP. Metal B stabilizes pyrophosphate, making it a better leaving group.

Several other residues are required for the proper orientation of the primer-template and dNTP in the polymerase active site. They impose steric and electrostatic constraints on nucleotide binding to increase the accuracy of replication. Contacts are formed between Lys522 and the alpha-phosphate, Tyr526 and His506 and the beta-phosphate, and Arg518 and the gamma-phosphate.

In addition, an essential contact is formed between His704 (the C-terminal residue) and the final phosphodiester bond on the primer strand. This contact is necessary to properly orient the primer for the addition of the next nucleotide.

In the protein, these sites are found in the fingers and palm.

Nucleotide Specificity:
Both A:T and G:C base pairs contain two hydrogen bond acceptors in their minor grooves. They are located at N3 for purines and O2 for pyrimidines. Arg429 and Gln615 each donate a hydrogen bond to these positions on the bases.

If a mismatch occurs, the spatial arrangement of the base pair is altered such that these contacts cannot form. As a result, polymerization slows down to allow the mismatch to be repaired by the exonuclease domain.

Exonuclease Activity:
The exonuclease active site is located approximately 35 angstroms from the polymerase active site. It also utilizes a two-metal mechanism (although only one metal is shown here). One of the metals stabilizes the formation of a hydroxide ion from water. The hydroxide ion acts as a nucleophile and attacks the phoshate group, which cleaves the phosphodiester bond. The second metal stabilizes the transition state and the oxyanion on the excised nucleotide.

Experimental results suggest that both metals are divalent, with Mn2+ and Zn2+ being the most likely substitutions. Only one of the metals is shown in this structure. It is forms a hydrogen bond with Asp5. The metal that is not shown forms hydrogen bonds with Asp5, Glu7, Asp174 and is located near the region outlined in green.

Summary:
The T7 replication complex is a member of the PolI family of DNA polymerases. Upon binding to thioredoxin, the polymerase is able to replicate the entire phage genome. Polymerization occurs via a two-metal mechanism, adding one dNTP and releasing pyrophosphate. If an incorrect nucleotide is incorporated into the DNA, contacts between the enzyme and the nucleotides are altered. This causes the rate of polymerization to slow, allowing the excision of the mismatch by the two-metal mechanism in the exonuclease site.