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The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication.

ABSTRACT: To better understand the energetics of accurate DNA replication, we directly measured ?G(o) for the incorporation of a nucleotide into elongating dsDNA in solution (?G(o)(incorporation)). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ??G(o)(incorporation) = 5.2 ± 1.34 kcal mol(-1)). Importantly, these direct measurements indicate that ??G(o)(incorporation) alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids; they may only have had to take advantage of the inherently more favorable ?G(o) for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.

SUBMITTER: Olson AC

PROVIDER: S-EPMC3561758 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication.

Olson Andrew C AC Patro Jennifer N JN Urban Milan M Kuchta Robert D RD

Journal of the American Chemical Society 20130117 4

To better understand the energetics of accurate DNA replication, we directly measured ΔG(o) for the incorporation of a nucleotide into elongating dsDNA in solution (ΔG(o)(incorporation)). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ΔΔG(o)(incorporation) = 5.2 ± 1.34 kcal mol(-1)). Importantly, these direct measurements indicate that ΔΔG(o)(incorporation) alone can account for th ...[more]

PMID: 23316816

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Project description:RNA polymerase (RNAP) is the primary machine responsible for transcription. Its ability to distinguish between correct (cognate) and incorrect (noncognate) nucleoside triphosphates (NTPs) is important for fidelity control in transcription. In this work, we investigated the substrate selection mechanism of T7 RNAP from the perspective of energetics. The dissociation free energies were determined for matched and unmatched base pairs in the preinsertion complex using the umbrella sampling method. A clear hydrogen-bond-rupture peak is observed in the potential of mean force curve for a matched base pair, whereas no such peaks are present in the position of mean force profiles for unmatched ones. The free-energy barrier could prevent correct substrates from being separated from the active site. Therefore, when NTPs diffuse into the active site, correct ones will stay for chemistry once they establish effective base pairing contacts with the template nucleotide, whereas incorrect ones will be withdrawn from the active site and rejected back to solution. This result provides an important energy evidence for the substrate selection mechanism of RNAP. Then we elucidated energetics and molecular details for correct NTP binding to the active site of the insertion complex. Our observations reveal that strong interactions act on the triphosphate of NTP to constrain its movement, whereas relatively weak interactions serve to position the base in the correct conformation. Triple interactions, hydrophobic contacts from residues M635 and Y639, base stacking from the 3' RNA terminal nucleotide, and base pairing from the template nucleotide act together to position the NTP base in a catalytically competent conformation. At last, we observed that incorrect NTPs cannot be as well-stabilized as the correct one in the active site when they are misincorporated in the insertion site. It is expected that our work can be helpful for comprehensively understanding details of this basic step in genetic transcription.

Dataset Information

The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication.

Publications

The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication.

Similar Datasets

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