biostudies-literatureChang CWelch FoundationNIGMS NIH HHSCancer Prevention and Research Institute of Texas2346https://www.ebi.ac.uk/biostudies/studies/S-EPMC9054841biostudies-literatureUnknown13(1)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.Nature communicationsIn crystallo observation of three metal ion promoted DNA polymerase misincorporation.PMC9054841RR190046C-2033-20200401T32 GM008280Lee Luo CGao YChang CfalseIn crystallo observation of three metal ion promoted DNA polymerase misincorporation.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.2022-01-01T00:00:00Z2022 Apr2026-05-31T06:06:11.119Z2025-02-19T01:55:24.463ZS-EPMC90548413548794710.1038/s41467-022-30005-3