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Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

Kuwahara M, Nagashima J, Hasegawa M, Tamura T, Kitagata R, Hanawa K, Hososhima S, Kasamatsu T, Ozaki H, Sawai H - Nucleic Acids Res. (2006)

Bottom Line: We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template.Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate.By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, Faculty of Engineering, Gunma University, Gunma 376-8515, Japan. kuwahara@chem.gunma-u.ac.jp

ABSTRACT
We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

Show MeSH
Decrease in the apparent relative catalytic efficiencies (kcat/Km)rel according to the numbers of modified dUs (t) on the primers and templates. Values were obtained from standing-start experiments using the following: P0–2, TA, TA6 and Vent(exo-) DNA polymerase (filled circles); PA, T0–3, dATP and Vent(exo-) DNA polymerase (open circles); P0–2, TA, TA6 and KOD(exo-) DNA polymerase (filled triangles); or PA, T0–3, dATP, and KOD(exo-) DNA polymerase (open triangles).
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fig5: Decrease in the apparent relative catalytic efficiencies (kcat/Km)rel according to the numbers of modified dUs (t) on the primers and templates. Values were obtained from standing-start experiments using the following: P0–2, TA, TA6 and Vent(exo-) DNA polymerase (filled circles); PA, T0–3, dATP and Vent(exo-) DNA polymerase (open circles); P0–2, TA, TA6 and KOD(exo-) DNA polymerase (filled triangles); or PA, T0–3, dATP, and KOD(exo-) DNA polymerase (open triangles).

Mentions: To determine the crucial step of the PCR, we carried out modified nucleotide standing-start experiments under conditions that achieve a single completed hit. Because the thermodynamic properties of oligonucleotides containing modified dU with (6-aminohexylamino)-2-oxoethyl group have been well-characterized (53), therefore we selected this type of a nucleotide for the experiments (Figure 2). The apparent values for Km and Vmax were obtained from the initial velocities of the standing-start primer extension reactions. The relative apparent reaction rate constant, (kcat)rel, is obtained by assuming that the Vmax value is proportional to total enzyme concentration and by setting the apparent relative reaction rate of a single incorporation of natural T to primer/template complex (P/T = P0/TA) at 1. The relative apparent catalytic efficiency, (kcat/Km)rel, is obtained by assuming that the apparent catalytic efficiency of a single natural T incorporation in P/T (P0/TA) is 1 (Table 3). Interestingly, the relative catalytic efficiency decreased at least 30-fold per modified dU residue when the modified dU was introduced in the growing terminus of the primer. On the other hand, it decreased only a few-fold per modified dU residue when the template contained modified dU (Table 3 and Figure 5). Of course, the apparent value for Km and Vmax obtained from this experiment can vary depending on the P/T sequence because the association–dissociation of the polymerase with P/T would influence the kinetic data; however, the apparent values of Km and Vmax for a single natural T incorporation in P/T (P0/TA) were almost equal to those of a single natural dA incorporation in P/T (PA/T0) under the same condition, indicating that the sequence difference in P/T would have little effect on the apparent value for Km and Vmax. Therefore, it appears that the decrease in relative apparent catalytic efficiency (kcat/Km)rel caused by the modified nucleotide on the primer and the template reflects the effects of the modified group on the true polymerization rate constant (kcat) and the rate constant for dissociation of enzyme from P/T (koff).


Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

Kuwahara M, Nagashima J, Hasegawa M, Tamura T, Kitagata R, Hanawa K, Hososhima S, Kasamatsu T, Ozaki H, Sawai H - Nucleic Acids Res. (2006)

Decrease in the apparent relative catalytic efficiencies (kcat/Km)rel according to the numbers of modified dUs (t) on the primers and templates. Values were obtained from standing-start experiments using the following: P0–2, TA, TA6 and Vent(exo-) DNA polymerase (filled circles); PA, T0–3, dATP and Vent(exo-) DNA polymerase (open circles); P0–2, TA, TA6 and KOD(exo-) DNA polymerase (filled triangles); or PA, T0–3, dATP, and KOD(exo-) DNA polymerase (open triangles).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636466&req=5

fig5: Decrease in the apparent relative catalytic efficiencies (kcat/Km)rel according to the numbers of modified dUs (t) on the primers and templates. Values were obtained from standing-start experiments using the following: P0–2, TA, TA6 and Vent(exo-) DNA polymerase (filled circles); PA, T0–3, dATP and Vent(exo-) DNA polymerase (open circles); P0–2, TA, TA6 and KOD(exo-) DNA polymerase (filled triangles); or PA, T0–3, dATP, and KOD(exo-) DNA polymerase (open triangles).
Mentions: To determine the crucial step of the PCR, we carried out modified nucleotide standing-start experiments under conditions that achieve a single completed hit. Because the thermodynamic properties of oligonucleotides containing modified dU with (6-aminohexylamino)-2-oxoethyl group have been well-characterized (53), therefore we selected this type of a nucleotide for the experiments (Figure 2). The apparent values for Km and Vmax were obtained from the initial velocities of the standing-start primer extension reactions. The relative apparent reaction rate constant, (kcat)rel, is obtained by assuming that the Vmax value is proportional to total enzyme concentration and by setting the apparent relative reaction rate of a single incorporation of natural T to primer/template complex (P/T = P0/TA) at 1. The relative apparent catalytic efficiency, (kcat/Km)rel, is obtained by assuming that the apparent catalytic efficiency of a single natural T incorporation in P/T (P0/TA) is 1 (Table 3). Interestingly, the relative catalytic efficiency decreased at least 30-fold per modified dU residue when the modified dU was introduced in the growing terminus of the primer. On the other hand, it decreased only a few-fold per modified dU residue when the template contained modified dU (Table 3 and Figure 5). Of course, the apparent value for Km and Vmax obtained from this experiment can vary depending on the P/T sequence because the association–dissociation of the polymerase with P/T would influence the kinetic data; however, the apparent values of Km and Vmax for a single natural T incorporation in P/T (P0/TA) were almost equal to those of a single natural dA incorporation in P/T (PA/T0) under the same condition, indicating that the sequence difference in P/T would have little effect on the apparent value for Km and Vmax. Therefore, it appears that the decrease in relative apparent catalytic efficiency (kcat/Km)rel caused by the modified nucleotide on the primer and the template reflects the effects of the modified group on the true polymerization rate constant (kcat) and the rate constant for dissociation of enzyme from P/T (koff).

Bottom Line: We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template.Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate.By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, Faculty of Engineering, Gunma University, Gunma 376-8515, Japan. kuwahara@chem.gunma-u.ac.jp

ABSTRACT
We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

Show MeSH