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A Short Adaptive Path from DNA to RNA Polymerases

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A Short Adaptive Path from DNA to RNA Polymerases A short adaptive path from DNA to RNA polymerases Christopher Cozensa, Vitor B. Pinheiroa, Alexandra Vaismanb, Roger Woodgateb, and Philipp Holligera,1 aMedical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; and bSection on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Edited by John Kuriyan, University of California, Berkeley, CA, and approved April 10, 2012 (received for review December 21, 2011) DNA polymerase substrate specificity is fundamental to genome long (14) and more commonly stall at +6–7 nt (14–16, 18) even integrity and to polymerase applications in biotechnology. In the after prolonged incubation. At the same time, there is compel- current paradigm, active site geometry is the main site of specificity ling structural and phylogenetic evidence for an adaptive path control. Here, we describe the discovery of a distinct specificity linking DNA to RNA polymerase activity in the evolution of the checkpoint located over 25 Å from the active site in the polymerase single-subunit RNA polymerases (ssRNAPs) of mitochondria thumb subdomain. In Tgo, the replicative DNA polymerase from and T-odd bacteriophages (e.g., T7 RNA polymerase), which are Thermococcus gorgonarius, we identify a single mutation (E664K) thought to derive from an ancestral polA-family DNA poly- within this region that enables translesion synthesis across a tem- merase (19–22). Thus, we (17) and others (6, 10, 16) have argued plate abasic site or a cyclobutane thymidine dimer. In conjunction that there must be a determinant of polymerase substrate spec- with a classic “steric-gate” mutation (Y409G) in the active site, ificity that has remained unidentified and that precludes syn- E664K transforms Tgo DNA polymerase into an RNA polymerase thesis of longer RNAs in the steric-gate mutants. capable of synthesizing RNAs up to 1.7 kb long as well as fully Here, we describe the discovery and characterization of a pseudouridine-, 5-methyl-C–,2′-fluoro–,or2′-azido–modified RNAs plausible candidate for such a specificity checkpoint. Using Tgo primed from a wide range of primer chemistries comprising DNA, DNA polymerase, the replicative DNA polymerase from Ther- RNA, locked nucleic acid (LNA), or 2′O-methyl–DNA. We find that mococcus gorgonarius, as our model system, we identify a region in E664K enables RNA synthesis by selectively increasing polymerase the thumb subdomain, and a single key residue (E664) within it. affinity for the noncognate RNA/DNA duplex as well as lowering Mutation of E664 to lysine (K) relieves the synthetic block for the Km for ribonucleotide triphosphate incorporation. This gate- RNA polymerization and, in the context of a steric-gate mutation keeper mutation therefore identifies a key missing step in the adap- (Y409G) and four previously described auxiliary mutations, ena- tive path from DNA to RNA polymerases and defines a previously bles the primer-dependent synthesis of long RNAs. Character- unknown postsynthetic determinant of polymerase substrate spec- ization of the phenotype suggests a molecular mechanism based ificity with implications for the synthesis and replication of noncog- on an enhanced primer/template duplex interaction interface. nate nucleic acid polymers. Results processivity | protein engineering | second gate Polymerase Region Enabling RNA Synthesis. Recent work in our group has focused on the engineering of polymerases for the eplicative polymerases require extraordinary specificity in synthesis and replication of unnatural nucleic acid polymers (23). Rsubstrate selection, incorporation, and replication both to This line of investigation led to the serendipitous identification of ensure fidelity and to exclude noncognate and/or damaged a polymerase (D4) with enhanced RNA polymerase activity, nucleotides from the genome. A particular threat to DNA ge- which is the starting point of the work described herein (Fig. S1A). nome integrity are ribonucleotide triphosphates (NTPs), which D4 derives from a variant of the replicative DNA polymerase of are present in the cell at concentrations up to 100-fold in excess the hyperthermophilic archaeon T. gorgonarius (Tgo) bearing of the cognate deoxyribonucleotide triphosphates (dNTPs) (1–3) additional mutations to disable uracil-stalling [V93Q (24)] and 3′- yet differ from them only by the presence of a 2′-hydroxyl(-OH) 5′ exonuclease (D141A and E143A) functions as well as the group. Indeed, although DNA polymerases have evolved to ex- “Therminator” (25) mutation (A485L), known to enhance in- clude NTPs from their active sites, incorporation does occur to corporation of unnatural substrates. This mutant polymerase a detectable degree, with significant implications for genome (henceforth termed TgoT) does not display RNA polymerase stability and repair (2, 4). This issue may be even more acute for activity above background levels (Fig. 1 and Fig. S1B): RNA thermophilic organisms, because high temperatures further in- synthesis by TgoT stalls after six to seven incorporations from crease genome instability by accelerating the spontaneous deg- a DNA primer. TgoT is also unable to extend an RNA primer radation of RNA (5). Control of NTP incorporation by DNA using NTPs. In contrast, D4 extends both DNA and RNA primers polymerases is therefore a paradigmatic case of the link between synthesizing RNAs of 20 nt under identical conditions. This gain polymerase substrate specificity and genome stability. of function in D4 is attributable to nine additional mutations, DNA polymerases from all three domains of life are known to comprising a cluster of eight mutations (P657T, E658Q, K659H, use a common strategy to prevent NTP incorporation into the Y663H, E664Q, D669A, K671N, and T676I) in the thumb sub- nascent strand, by exerting stringent geometric control of the domain and a single mutation (L403P) in the A-motif (Fig. S1D). chemical nature of the 2′ position of the incoming nucleotide Having identified mutations that enhance RNA polymerase through a single active site residue, the “steric gate” (6). This activity, we sought to dissect their contribution to the phenotype strategy is so efficient that mutation of the steric gate alone (e.g., in the context of a more permissive active site for RNA synthesis. to an amino acid with a smaller side chain) can reduce dis- BIOCHEMISTRY crimination against NTP incorporation by several orders of mag- nitude (6–13). However, although steric-gate mutations generally Author contributions: P.H. designed research; C.C., V.B.P., and A.V. performed research; render DNA polymerases permissive for NTP incorporation, C.C., V.B.P., A.V., R.W., and P.H. analyzed data; and C.C. and P.H. wrote the paper. they do not by themselves enable synthesis of RNAs beyond The authors declare no conflict of interest. short termination products. Indeed, engineering efforts using This article is a PNAS Direct Submission. rational design (9), in vitro and in vivo screening (14, 15), or Freely available online through the PNAS open access option. directed evolution by phage display (16) and compartmentalized 1To whom correspondence should be addressed. E-mail: [email protected]. self-replication (17) have so far only yielded polymerases that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. can synthesize RNAs up to 58 nucleotide incorporations (nt) 1073/pnas.1120964109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1120964109 PNAS | May 22, 2012 | vol. 109 | no. 21 | 8067–8072 Downloaded by guest on September 29, 2021 91 95 402 410 483 487 655 678 A TgoT QDQPA ... YLDFRSLYP ... QRLIK ... VPPEKLVIYEQITRDLKDYKATGP D4 QDQPA ... YPDFRSLYP ... QRLIK ... VPTQHLVIHQQITRALNDYKAIGP TGE QDQPA ... YLDFRSLGP ... QRLIK ... VPPEKLVIYEQITRDLKDYKATGP TYK QDQPA ... YLDFRSLYP ... QRLIK ... VPPEKLVIYKQITRDLKDYKATGP TGK QDQPA ... YLDFRSLGP ... QRLIK ... VPPEKLVIYKQITRDLKDYKATGP BDSteric gate A485L V93Q C mutation TGK (Y409G) Primer only TgoT TGE TGK 0.5 m 10 m 1 m 5 m TYK +74nt +74nt Primer Template exonuclease mutations (D141A, E143A) RNA Second gate RNA primer mutation (E664K) primer Fig. 1. Mutations enabling RNA synthesis. (A) Sequence locations of mutations present in D4, the engineered variant TGK (TgoT: Y409G E664K), and in- termediate TGE and TYK polymerases are shown together with the parent TgoT. (B) TGK mutations are mapped onto the structure of a secondary complex of the closely related Pfu polymerase [Protein Data Bank (PDB) ID code 4AIL]. The steric-gate mutation and second-gate mutation are shown in red, secondary mutations present in the starting polymerase TgoT (uracil stalling function and Therminator mutation) are shown in orange, and exonuclease mutations are shown in yellow. (C) RNA polymerase activity of polymerases TgoT, TGE, TYK, and TGK (from an RNA primer). (D) Time course of synthesis of E. coli tRNATyr from RNA primers, showing the appearance of the full-length product (+74 nt) within 30 s for TGK. Previous work on the polB-family polymerases had identified mutation of E664 is both necessary and sufficient for efficient a conserved tyrosine (Tgo: Y409) as the steric-gate residue. RNA synthesis in conjunction with a steric-gate mutation. Mutation of Y409 to a smaller side-chain amino acid was found Having established a key role for E664 in enabling RNA syn- to reduce NTP/dNTP discrimination by more than 103-fold, yet it thesis, we sought to identify optimal mutations of this residue for is not sufficient
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