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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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(A) Library of non-natural nucleotides used to interrogate the mechanism accounting for the preferential utilization of dATP during the replication of an abasic site. For clarity, only the nucleobases of the 5-substituted indolyl nucleotides are shown. (B) SAR highlighting the importance of π-electron surface area of the incoming nucleotide for efficient incorporation opposite an abasic site. See text for details.
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Figure 1: (A) Library of non-natural nucleotides used to interrogate the mechanism accounting for the preferential utilization of dATP during the replication of an abasic site. For clarity, only the nucleobases of the 5-substituted indolyl nucleotides are shown. (B) SAR highlighting the importance of π-electron surface area of the incoming nucleotide for efficient incorporation opposite an abasic site. See text for details.

Mentions: One commonly formed DNA lesion is an abasic site that, due to the lack of coding information, is highly pro-mutagenic (9). Although abasic sites are non-instructional, high-fidelity DNA polymerases such as the bacteriophage T4 DNA polymerase preferentially utilize dATP (10). This phenomenon is commonly referred to as the ‘A-rule’ of translesion DNA synthesis (11–18). While the mutagenic consequences of the ‘A-rule’ are well documented (19–21), the mechanistic basis for the preferential utilization of dATP has not been firmly established. Our investigations of the ‘A-rule’ have focused on characterizing the kinetic behavior of the library of non-natural nucleotides illustrated in Figure 1A during insertion opposite this DNA lesion (22–28). While these analogs mimic the core structure of dATP, the introduction of various functional groups to the 5-position of the indole ring provides a way to systematically modulate biophysical features such as shape, size, hydrophobicity, dipole moment and π-electron surface area. The kinetic parameters for their utilization were used to develop a structure–activity relationship (SAR) defining key biophysical features required for optimal incorporation opposite an abasic site (Figure 1B) (23). The parabolic nature of this plot reveals that the catalytic efficiency (kpol/Kd, app) for nucleotide incorporation is dependent upon an optimal π-electron surface area that most closely mimics that of a natural Watson–Crick base pair.Figure 1.


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

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

(A) Library of non-natural nucleotides used to interrogate the mechanism accounting for the preferential utilization of dATP during the replication of an abasic site. For clarity, only the nucleobases of the 5-substituted indolyl nucleotides are shown. (B) SAR highlighting the importance of π-electron surface area of the incoming nucleotide for efficient incorporation opposite an abasic site. See text for details.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045600&req=5

Figure 1: (A) Library of non-natural nucleotides used to interrogate the mechanism accounting for the preferential utilization of dATP during the replication of an abasic site. For clarity, only the nucleobases of the 5-substituted indolyl nucleotides are shown. (B) SAR highlighting the importance of π-electron surface area of the incoming nucleotide for efficient incorporation opposite an abasic site. See text for details.
Mentions: One commonly formed DNA lesion is an abasic site that, due to the lack of coding information, is highly pro-mutagenic (9). Although abasic sites are non-instructional, high-fidelity DNA polymerases such as the bacteriophage T4 DNA polymerase preferentially utilize dATP (10). This phenomenon is commonly referred to as the ‘A-rule’ of translesion DNA synthesis (11–18). While the mutagenic consequences of the ‘A-rule’ are well documented (19–21), the mechanistic basis for the preferential utilization of dATP has not been firmly established. Our investigations of the ‘A-rule’ have focused on characterizing the kinetic behavior of the library of non-natural nucleotides illustrated in Figure 1A during insertion opposite this DNA lesion (22–28). While these analogs mimic the core structure of dATP, the introduction of various functional groups to the 5-position of the indole ring provides a way to systematically modulate biophysical features such as shape, size, hydrophobicity, dipole moment and π-electron surface area. The kinetic parameters for their utilization were used to develop a structure–activity relationship (SAR) defining key biophysical features required for optimal incorporation opposite an abasic site (Figure 1B) (23). The parabolic nature of this plot reveals that the catalytic efficiency (kpol/Kd, app) for nucleotide incorporation is dependent upon an optimal π-electron surface area that most closely mimics that of a natural Watson–Crick base pair.Figure 1.

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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