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Quantifying the energetic contributions of desolvation and π-electron density during translesion DNA synthesis.

Motea EA, Lee I, Berdis AJ - Nucleic Acids Res. (2010)

Bottom Line: In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density.We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site.Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.

ABSTRACT
This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2'-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the K(d, app) values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

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Measuring extension beyond an abasic site. (A) Experiments were performed under single-turnover conditions in which 500 nM bacteriophage T4 DNA polymerase was incubated with 250 nM 13/20SP-mer in assay buffer containing 10 mM Mg2+ and mixed with 150 µM dXTP for 5 min. To initiate extension beyond the lesion, 500 µM dGTP, the correct nucleotide for the next three insertion positions, was added and the reactions were quenched with 350 mM EDTA at 180 s. (B) Denaturing gel electrophoresis data for the incorporation and extension of nucleotides opposite an abasic site catalyzed by the bacteriophage T4 DNA polymerase. Only nucleotides containing hydrogen-bonding functional groups (dATP, 5-CITP and 5-MeCITP) showed extension beyond the abasic site. (C) Quantification of gel electrophoresis data provided in panel B. Blue bars represent percentage of 14-mer product (incorporation of nucleotide opposite abasic site) while red bars represent percentage of 14-mer product that is elongated after addition of dGTP.
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Figure 3: Measuring extension beyond an abasic site. (A) Experiments were performed under single-turnover conditions in which 500 nM bacteriophage T4 DNA polymerase was incubated with 250 nM 13/20SP-mer in assay buffer containing 10 mM Mg2+ and mixed with 150 µM dXTP for 5 min. To initiate extension beyond the lesion, 500 µM dGTP, the correct nucleotide for the next three insertion positions, was added and the reactions were quenched with 350 mM EDTA at 180 s. (B) Denaturing gel electrophoresis data for the incorporation and extension of nucleotides opposite an abasic site catalyzed by the bacteriophage T4 DNA polymerase. Only nucleotides containing hydrogen-bonding functional groups (dATP, 5-CITP and 5-MeCITP) showed extension beyond the abasic site. (C) Quantification of gel electrophoresis data provided in panel B. Blue bars represent percentage of 14-mer product (incorporation of nucleotide opposite abasic site) while red bars represent percentage of 14-mer product that is elongated after addition of dGTP.

Mentions: We previously demonstrated that the bacteriophage T4 DNA polymerase extends beyond dATP or dGTP when paired opposite an abasic site (10,26). However, this high-fidelity DNA polymerase cannot extend beyond any of the 5-substituted indolyl nucleotides illustrated in Figure 1A. The lack of extension could be caused by alterations in the conformation of the primer template (43) or by a lack of hydrogen-bonding functional groups that serve as recognition elements for elongation (44). To differentiate between these models, we used the experimental protocol outlined in Figure 3A to quantify extension beyond the non-natural nucleotides developed in this study. These experiments employed single-turnover conditions in which DNA polymerase (500 nM) and DNA substrate (250 nM) were mixed with 150 µM non-natural nucleotide and 10 mM Mg2+ to allow for incorporation opposite the abasic site. After four half-lives (time required to obtain 95% mispair formation), 500 µM dGTP was added to initiate elongation beyond the abasic site. Aliquots were quenched with EDTA at variable times and analyzed via denaturing gel electrophoresis to visualize product formation. Representative data provided in Figure 3B shows that the T4 DNA polymerase does not extend beyond hydrophobic nucleotides such as 5-NITP, 5-F5-PhITP, 5-F4OMe-PhITP and 5-PhITP since only 14-mer product is observed (Figure 4B, lanes 2, 5, 6 and 7, respectively). In contrast, the T4 DNA polymerase extends beyond 5-CITP (lane 3) and 5-MeCITP (lane 4) almost as efficiently as dATP (lane 8). In fact, quantifying the amount of primer extension reveals that the efficiency for extending beyond 5-CITP and 5-MeCITP is only 20% lower than that for dATP (Figure 3C). These data support a mechanism in which the presence of functional groups that can participate in hydrogen-bonding interactions is an important determinant for elongation.Figure 3.


Quantifying the energetic contributions of desolvation and π-electron density during translesion DNA synthesis.

Motea EA, Lee I, Berdis AJ - Nucleic Acids Res. (2010)

Measuring extension beyond an abasic site. (A) Experiments were performed under single-turnover conditions in which 500 nM bacteriophage T4 DNA polymerase was incubated with 250 nM 13/20SP-mer in assay buffer containing 10 mM Mg2+ and mixed with 150 µM dXTP for 5 min. To initiate extension beyond the lesion, 500 µM dGTP, the correct nucleotide for the next three insertion positions, was added and the reactions were quenched with 350 mM EDTA at 180 s. (B) Denaturing gel electrophoresis data for the incorporation and extension of nucleotides opposite an abasic site catalyzed by the bacteriophage T4 DNA polymerase. Only nucleotides containing hydrogen-bonding functional groups (dATP, 5-CITP and 5-MeCITP) showed extension beyond the abasic site. (C) Quantification of gel electrophoresis data provided in panel B. Blue bars represent percentage of 14-mer product (incorporation of nucleotide opposite abasic site) while red bars represent percentage of 14-mer product that is elongated after addition of dGTP.
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Figure 3: Measuring extension beyond an abasic site. (A) Experiments were performed under single-turnover conditions in which 500 nM bacteriophage T4 DNA polymerase was incubated with 250 nM 13/20SP-mer in assay buffer containing 10 mM Mg2+ and mixed with 150 µM dXTP for 5 min. To initiate extension beyond the lesion, 500 µM dGTP, the correct nucleotide for the next three insertion positions, was added and the reactions were quenched with 350 mM EDTA at 180 s. (B) Denaturing gel electrophoresis data for the incorporation and extension of nucleotides opposite an abasic site catalyzed by the bacteriophage T4 DNA polymerase. Only nucleotides containing hydrogen-bonding functional groups (dATP, 5-CITP and 5-MeCITP) showed extension beyond the abasic site. (C) Quantification of gel electrophoresis data provided in panel B. Blue bars represent percentage of 14-mer product (incorporation of nucleotide opposite abasic site) while red bars represent percentage of 14-mer product that is elongated after addition of dGTP.
Mentions: We previously demonstrated that the bacteriophage T4 DNA polymerase extends beyond dATP or dGTP when paired opposite an abasic site (10,26). However, this high-fidelity DNA polymerase cannot extend beyond any of the 5-substituted indolyl nucleotides illustrated in Figure 1A. The lack of extension could be caused by alterations in the conformation of the primer template (43) or by a lack of hydrogen-bonding functional groups that serve as recognition elements for elongation (44). To differentiate between these models, we used the experimental protocol outlined in Figure 3A to quantify extension beyond the non-natural nucleotides developed in this study. These experiments employed single-turnover conditions in which DNA polymerase (500 nM) and DNA substrate (250 nM) were mixed with 150 µM non-natural nucleotide and 10 mM Mg2+ to allow for incorporation opposite the abasic site. After four half-lives (time required to obtain 95% mispair formation), 500 µM dGTP was added to initiate elongation beyond the abasic site. Aliquots were quenched with EDTA at variable times and analyzed via denaturing gel electrophoresis to visualize product formation. Representative data provided in Figure 3B shows that the T4 DNA polymerase does not extend beyond hydrophobic nucleotides such as 5-NITP, 5-F5-PhITP, 5-F4OMe-PhITP and 5-PhITP since only 14-mer product is observed (Figure 4B, lanes 2, 5, 6 and 7, respectively). In contrast, the T4 DNA polymerase extends beyond 5-CITP (lane 3) and 5-MeCITP (lane 4) almost as efficiently as dATP (lane 8). In fact, quantifying the amount of primer extension reveals that the efficiency for extending beyond 5-CITP and 5-MeCITP is only 20% lower than that for dATP (Figure 3C). These data support a mechanism in which the presence of functional groups that can participate in hydrogen-bonding interactions is an important determinant for elongation.Figure 3.

Bottom Line: In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density.We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site.Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.

ABSTRACT
This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2'-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the K(d, app) values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

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