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Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases

Abstract : Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2' modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.
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https://hal-pasteur.archives-ouvertes.fr/pasteur-03086156
Contributor : Marie de Tarragon <>
Submitted on : Tuesday, December 22, 2020 - 12:32:49 PM
Last modification on : Tuesday, January 5, 2021 - 3:40:37 AM

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Kelly Zatopek, Ece Alpaslan, Thomas Evans, Ludovic Sauguet, Andrew Gardner. Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases. Nucleic Acids Research, Oxford University Press, 2020, 48 (21), pp.12204-12218. ⟨10.1093/nar/gkaa986⟩. ⟨pasteur-03086156⟩

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