In crystallo observation of three metal ion promoted DNA polymerase misincorporation

By Caleb Chang, Christie Lee Luo, Yang Gao. 2022

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Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis. By observing DNA polymerase misincorporation with time-resolved crystallography, the authors visualize three-metal ion dependent polymerase catalysis and identify A-site metal-mediated primer alignment as a key step in nucleotide discrimination.

Subfields: Genetics, Molecular Biology

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