title: A short adaptive path from DNA to RNA polymerases.
abstract: DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identify a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic "steric-gate" mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long as well as fully pseudouridine-, 5-methyl-C-, 2'-fluoro-, or 2'-azido-modified RNAs primed from a wide range of primer chemistries comprising DNA, RNA, locked nucleic acid (LNA), or 2'O-methyl-DNA. We find that E664K enables RNA synthesis by selectively increasing polymerase affinity for the noncognate RNA/DNA duplex as well as lowering the K(m) for ribonucleotide triphosphate incorporation. This gatekeeper mutation therefore identifies a key missing step in the adaptive path from DNA to RNA polymerases and defines a previously unknown postsynthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of noncognate nucleic acid polymers.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH,  United Kingdom.
authors.forename: Christopher
authors.initials: C
authors.lastname: Cozens
authors.forename: Vitor B
authors.initials: VB
authors.lastname: Pinheiro
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vaisman
authors.forename: Roger
authors.initials: R
authors.lastname: Woodgate
authors.forename: Philipp
authors.initials: P
authors.lastname: Holliger
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D019641:DNA, Archaeal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase