Download MendeleyMultiple deprotonation paths of the nucleophile 3'-OH in the DNA synthesis reaction.
Gregory, M.T., Gao, Y., Cui, Q., Yang, W.(2021) Proc Natl Acad Sci U S A 118
- PubMed: 34088846
- DOI: https://doi.org/10.1073/pnas.2103990118
- Primary Citation of Related Structures:
7M7L, 7M7M, 7M7N, 7M7O, 7M7P, 7M7Q, 7M7R, 7M7S, 7M7T, 7M7U, 7M7Y, 7M7Z, 7M80, 7M81, 7M82, 7M83, 7M84, 7M85, 7M86, 7M87, 7M88, 7M89, 7M8A, 7M8B, 7M8C, 7M8D - PubMed Abstract:
DNA synthesis by polymerases is essential for life. Deprotonation of the nucleophile 3'-OH is thought to be the obligatory first step in the DNA synthesis reaction. We have examined each entity surrounding the nucleophile 3'-OH in the reaction catalyzed by human DNA polymerase (Pol) ¦Ç and delineated the deprotonation process by combining mutagenesis with steady-state kinetics, high-resolution structures of in crystallo reactions, and molecular dynamics simulations. The conserved S113 residue, which forms a hydrogen bond with the primer 3'-OH in the ground state, stabilizes the primer end in the active site. Mutation of S113 to alanine destabilizes primer binding and reduces the catalytic efficiency. Displacement of a water molecule that is hydrogen bonded to the 3'-OH using the 2'-OH of a ribonucleotide or 2'-F has little effect on catalysis. Moreover, combining the S113A mutation with 2'-F replacement, which removes two potential hydrogen acceptors of the 3'-OH, does not reduce the catalytic efficiency. We conclude that the proton can leave the O3' via alternative paths, supporting the hypothesis that binding of the third Mg 2+ initiates the reaction by breaking the ¦Á-¦Â phosphodiester bond of an incoming deoxyribonucleoside triphosphate (dNTP).
Organizational Affiliation:
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892.
