T7 DNA polymerase

T7 DNA polymerase is a heterodimeric 92-kDa enzyme composed of the phage gene 5 protein and the host-encoded thioredoxin in a 1 1 stoichiometry. In addition to the highly processive polymerase activity, the native T7 DNA Pol has a strong 

3 — 5 -exonuclease activity specific for both ssDNA and dsDNA. No 5 -nuclease activity has been found with T7 DNA Pol. Modified forms of T7 DNA Pol from which the 3 — 5 -exonuclease activity has been removed are commercially available. 

The following reaction conditions are typically used for second-strand DNA synthesis on ssDNA templates 40 mM Tris—Cl (pH 7,5), 15 mM MgCl2,25 mM NaCl, 5 mM DTT, 0.25 mM each of four dNTPs, 1 /ig template (plus primer) DNA, and 0.5-1 U of polymerase in a 20-(ul reaction volume. The mixture is incubated at room temperature or 37°C for 1 hr. The reaction can be stopped by adding excess EDTA or by heating at 75°C for 10 min. 

Unit definition One polymerase unit is the amount of enzyme that incorporates 10 nmol of dNTPs into acid-insoluble products in 30 min. 

Unit definition One exonuclease unit catalyzes the release of 10 nmol of 

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Synthesize second strand with T7 DNA polymerase and T4 ligase... 

Tabor, S., and Richardson, C.C. (1987) Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase./. Biol. Chem. 262, 15330-15333. 

A sample of double-stranded DNA is denatured. One of the resulting single strands is used as a template to direct the synthesis of a complementary strand of radioactive DNA using a suitable DNA polymerase. The "Klenow fragment" of E. coli, DNA polymerase I, reverse transcriptase from a retrovirus, bacteriophage T7 DNA polymerase, Taq polymerase, and specially engineered enzymes produced from cloned genes have all been used. 

Because step 3 will be discussed in great detail in the sections below, it is appropriate to provide a brief summary of how it was discovered, using the T7 DNA polymerase as an example (Donlin et al, 1991 Patel et al., 1991 Wong et al., 1991). The rate-limiting step was observed when the dependence of the burst amplitude (corresponding to the incorporation of the first nucleotide) on nucleotide concentration was examined. This experiment provided kpol of step 3 (Fig. 1), which was lower than all the other... 

It has been shown for the Klenow fragment that the PPi product has only a fivefold lower affinity for the E p/t complex than dNTPs, suggesting that the product of the reaction could compete for binding of dNTPs (Kuchta et al., 1987). However, for T7 DNA polymerase, the affinity of PPi is extremely low and is nowhere near comparable to affinities for correct nucleotide binding, differing by nearly a factor of 1000 (Patel et al, 1991). T4 has similarly reduced affinities for PPi compared with Klenow, being in the low millimolar range (Capson et al, 1992). 

Florian J, MF Goodman, A Warshel (2003) Computer simulation of the chemical catalysis of DNA polymerases Discriminating between alternative nucleotide insertion mechanisms for T7 DNA polymerase. J. Am. Chem. Soc. 125 (27) 8163—8177... 

Thermus aquaticus Thermus thermophilus Thermus filiformis Deinococcus radiodurans Escherichia coli Haemophilus influenzae Streptococus pneumoniae Mycobacterium tuberculosis Mycobacterium laprae Treponema pallidum Chlamydia trachomatis Boriela burgdorferi Helicobacter pyroli Lactococcus lactis Mythelobacterium Rhodothermus obamensis Rickettsia prowazekii Streptomyces coelicolor Bacillus stearothermophilus Synechocystis sp Aquifex aeolicus Apse-1 DNA polymerase T7 DNA polymerase T5 DNA polymerase ... 

Except for the altered forms of T7 DNA polymerase and Taq DNA polymerase (23), all of the DNA polymerases identified above contain a 3 - 5 exonuclease function that specifically degrades single-stranded DNA to produce 5 -nucleo-side monophosphates. In vivo, the 3 - 5 exonuclease functions during DNA synthesis to hydrolyze misincorpo rated nucleotides at the 3 -terminus of a growing DNA chain, and in doing so contributes as much as a factor of 100 to the overall fidelity of DNA replication (24, 25). 

Figure 1 Mechanism of DNA polymerization, (a) The structure of T7 DNA polymerase in a complex with DNA and an incoming nucleotide is shown with a fluorescent label attached to C514. Changes in the fluorescence allow quantification of the nucleotide-induced change in structure and its role in selectivity. Residues 233-411 and 436-454 have been removed to reveal the active site. Shown also are the O-helix and key catalytic residues From PDB 1 T7P (17). (b) The time dependence of the fluorescence change induced by nucleotide binding is shown at three concentrations of dCTP. The inset shows the measurement of the rate of dCTP dissociation from the E.DNAdd-dNTP complex. Analysis of these data defined the role of enzyme conformational changes in nucleotide selectivity. Both figures are reproduced with permission from Reference 6.

The molecular ruler methodology for determining the size of the active site of DNA polymerase was also applied to the investigations of other DNA polymerases, such as T7 DNA polymerase and Dpo4 polymerase [60, 61]. 

Kim, T. W., Brieba, L. G., Ellenberger, T. and Kool, E. T. (2006) Functional evidence for a small and rigid active site in a high fidelity DNA polymerase probing T7 DNA polymerase with variably sized base pairs. J. Biol. Chem., 281, 2289-2295. 

Aliquots from the plasmid DNA of 6 pg for T7 DNA polymerase sequencing or 500 ng for cycle sequencing were prepared. 

To the annealed oligonucleotide/template mixture, add 10pL 10mM ATP, lOpL 25mM dNTPs, 15pL lOOmM DTT, 30 Weiss units T4 DNA ligase, 30 units T7 DNA polymerase. 

J., Richardson, C.C., Romano, L.J., and Ellenberger, T. (2004) Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis. Proc. Natl. Acad. Sci. USA, 101, 16186-16191. 

Undsley, J.E. and Fuchs, R.P. (1994) Use of single-turnover kinetics to study bulky adduct bypass by T7 DNA polymerase. Biochemistry, 33, 764—772. 

Furge, L.L. and Guengerich, F.P. (1998) Pre-steady-state kinetics of nucleotide insertion following 8-oxo-7,8-dihydro-guanine base pair mismatches by bacteriophage T7 DNA polymerase exo. Biochemistry, 37, 3567-3574. 

Zang, H., Harris, T.M., and Guengerich, F.P. (2005) Kinetics of nucleotide incorporation opposite DNA bulky guanine N2 adducts by processive bacteriophage T7 DNA polymerase (exonuclease-) and HIV-1 reverse... 

Wang, L., Broyde, S., and Zhang, Y. (2009) Polymerase-tailored variations in the water-mediated and substrate-assisted mechanism for nucleotidyl transfer insights from a study of T7 DNA polymerase./. Mol. Biol, 389, 787-796. 

The quahty of the purified product is an important aspect, and must be ensured because it can influence the potency of dSLIM. Therefore, integrity is tested via exonuclease activity of T7-DNA-polymerase degrading residual DNA molecules with open ends and subsequent gel electrophoresis. Furthermore, the content of endotoxin is determined by an end-point Limulus amoebocyte lysate (LAL) test, and must meet strict standards (< 10 EU mg DNA). 

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