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Induced Fit in the Selection of Correct versus Incorrect Nucleotides by DNA Polymerase β.

ABSTRACT: DNA polymerase β (Pol β) repairs single-nucleotide gapped DNA (sngDNA) by enzymatic incorporation of the Watson-Crick partner nucleotide at the gapped position opposite the templating nucleotide. The process by which the matching nucleotide is incorporated into a sngDNA sequence has been relatively well-characterized, but the process of discrimination from nucleotide misincorporation remains unclear. We report here NMR spectroscopic characterization of full-length, uniformly labeled Pol β in apo, sngDNA-bound binary, and ternary complexes containing matching and mismatching nucleotide. Our data indicate that, while binding of the correct nucleotide to the binary complex induces chemical shift changes consistent with the process of enzyme closure, the ternary Pol β complex containing a mismatching nucleotide exhibits no such changes and appears to remain in an open, unstable, binary-like conformation. Our findings support an induced-fit mechanism for polymerases in which a closed ternary complex can only be achieved in the presence of matching nucleotide.

SUBMITTER: Moscato B

PROVIDER: S-EPMC8259413 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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once they establish effective base pairing contacts with the template nucleotide, whereas incorrect ones will be withdrawn from the active site and rejected back to solution.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.

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Induced Fit in the Selection of Correct versus Incorrect Nucleotides by DNA Polymerase β.

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