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Building better polymerases: Engineering the replication of expanded genetic alphabets

Open AccessPublished:October 01, 2020DOI:https://doi.org/10.1074/jbc.REV120.013745
      DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.
      DNA polymerases (
      • Goodman M.
      • Tippin B.
      The expanding polymerase universe.
      ), RNA polymerases (
      • Young R.A.
      RNA polymerase II.
      ), and reverse transcriptases (
      • Autexier C.
      • Lue N.F.
      The structure and function of telomerase reverse transcriptase.
      ) are enzymes central to terran biology and permit life to propagate and exploit its genetic inheritance. DNA polymerases are also workhorses in biotechnology (
      • Aschenbrenner J.
      • Marx A.
      DNA polymerases and biotechnological applications.
      ,
      • Gardner A.F.
      • Jackson K.M.
      • Boyle M.M.
      • Buss J.A.
      • Potapov V.
      • Gehring A.M.
      • Zatopek K.M.
      • Corrêa Jr., I.R.
      • Ong J.L.
      • Jack W.E.
      Therminator DNA polymerase: modified nucleotides and unnatural substrates.
      ), where they often must manage DNA molecules having unusual structural features. For example, some WT bacterial polymerases can replicate DNA-containing UBPs that interact by steric complementarity without any interbase hydrogen bonding (
      • Betz K.
      • Malyshev D.A.
      • Lavergne T.
      • Welte W.
      • Diederichs K.
      • Dwyer T.J.
      • Ordoukhanian P.
      • Romesberg F.E.
      • Marx A.
      Klentaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry.
      ) (Fig. 1). This finding has been exploited to obtain engineered strains of Escherichia coli that can express proteins built from noncanonical amino acids with improved efficiency (
      • Fischer E.C.
      • Hashimoto K.
      • Zhang Y.
      • Feldman A.W.
      • Dien V.T.
      • Karadeema R.J.
      • Adhikary R.
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      • Romesberg F.E.
      New codons for efficient production of unnatural proteins in a semisynthetic organism.
      ,
      • Liu C.C.
      • Schultz P.G.
      Adding new chemistries to the genetic code.
      ). This capability was a driver of the recent $2.5 billion acquisition of Synthorx by Sanofi-Aventis.
      Figure thumbnail gr1
      Figure 1Chemical structures of nucleobase pairs. R indicates the point of covalent attachment to either deoxyribose or ribose in DNA or RNA, respectively. A, Watson–Crick nucleobase pairs (
      • Watson J.D.
      • Crick F.H.C.
      Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid.
      ). B, isosteric UBPs developed by Kool and co-workers (
      • Kool E.T.
      Replacing the nucleobases in DNA with designer molecules.
      ). C, hydrophobic UBPs developed by Romesberg and co-workers (
      • Feldman A.W.
      • Romesberg F.E.
      Expansion of the genetic alphabet: a chemist's approach to synthetic biology.
      ,
      • Malyshev D.A.
      • Romesberg F.E.
      The expanded genetic alphabet.
      ). D, hydrogen-bonding UBPs used in hachimoji DNA developed by Benner and co-workers (
      • Piccirilli J.A.
      • Krauch T.
      • Moroney S.E.
      • Benner S.A.
      Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet.
      ,
      • Benner S.A.
      • Karalkar N.B.
      • Hoshika S.
      • Laos R.
      • Shaw R.W.
      • Matsuura M.
      • Fajardo D.
      • Moussatche P.
      Alternative Watson-Crick synthetic genetic systems.
      ). E, hydrophobic UBPs developed by Hirao and co-workers (
      • Hirao I.
      • Kimoto M.
      • Yamashige R.
      Natural versus artificial creation of base pairs in DNA: origin of nucleobases from the perspective of unnatural base pair studies.
      ,
      • Yamashige R.
      • Kimoto M.
      • Takezawa Y.
      • Sato A.
      • Mitsui T.
      • Yokoyama S.
      • Hirao I.
      Highly specific unnatural base pair systems as a third base pair for PCR amplification.
      ), where R1 can be a variety of substituents.
      DNA polymerase variants are also playing key roles at other scientific frontiers. For example, NextGen sequencing (
      • Metzker M.L.
      Sequencing technologies—the next generation.
      ) has transformed medicine (
      • McCombie W.R.
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      Next-generation sequencing technologies.
      ), but only because it was enabled by the use of DNA polymerase variants capable of replicating “Watson–Crick” (WC) deoxynucleoside triphosphates (
      • Watson J.D.
      • Crick F.H.C.
      Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid.
      ) “tagged” with fluorescent markers and carrying blocking groups appended to their 3′-positions (
      • Leary J.J.
      • Brigati D.J.
      • Ward D.C.
      Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.
      ).

      Expanded genetic alphabets and engineered DNA polymerases

      These prior studies, however, only “scratch the surface” of the possible modified nucleotides that have scientific, biotechnological, and medical value. For example, DNA can have many more independently replicable groups than A, T, G, or C (Fig. 1). Scientifically, these groups can be varied to learn whether WC DNA represents the only molecular solution for the long-term storage and replication of biological information (
      • Piccirilli J.A.
      • Krauch T.
      • Moroney S.E.
      • Benner S.A.
      Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet.
      ,
      • Orgel L.E.
      Molecular replication.
      ,
      • Robertson M.P.
      • Joyce G.F.
      The origins of the RNA world.
      ,
      • Powner M.W.
      • Gerland B.
      • Sutherland J.D.
      Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions.
      ). Alexander Rich was likely the first to recognize the importance of this question, suggesting in 1962 that the isocytidine:isoguanine (S:B) pair (Fig. 1) might be used as an additional information storing unit in DNA (
      • Rich A.
      Problems of evolution and biochemical information transfer.
      ).
      Further, many have asked whether the sugar and phosphate components of the DNA backbone might be modified. Synthetic biologists have therefore synthesized numerous such modified DNA molecules to delineate molecular changes that can (and cannot) be tolerated by duplex DNA (
      • Herdewijn P.
      Nucleic acids with a six-membered carbohydrate mimic in the backbone.
      ,
      • Taylor A.I.
      • Houlihan G.
      • Holliger P.
      Beyond DNA and RNA: the expanding toolbox of synthetic genetics.
      ,
      • Hutter D.
      • Blaettler M.O.
      • Benner S.A.
      From phosphate to bis(methylene)sulfone: non-ionic backbone linkers.
      ).
      The extent to which the core concept of WC pairing can be changed has also been explored by synthetic biologists. For example, the laboratories of Eric Kool (
      • Kool E.T.
      Replacing the nucleobases in DNA with designer molecules.
      ), Floyd Romesberg (
      • Feldman A.W.
      • Romesberg F.E.
      Expansion of the genetic alphabet: a chemist's approach to synthetic biology.
      ,
      • Malyshev D.A.
      • Romesberg F.E.
      The expanded genetic alphabet.
      ), and Ichiro Hirao (
      • Hirao I.
      • Kimoto M.
      • Yamashige R.
      Natural versus artificial creation of base pairs in DNA: origin of nucleobases from the perspective of unnatural base pair studies.
      ,
      • Yamashige R.
      • Kimoto M.
      • Takezawa Y.
      • Sato A.
      • Mitsui T.
      • Yokoyama S.
      • Hirao I.
      Highly specific unnatural base pair systems as a third base pair for PCR amplification.
      ) have attempted to dispense with interbase hydrogen bonding (Fig. 1).
      Developing a different approach, Steven Benner and co-workers (
      • Piccirilli J.A.
      • Krauch T.
      • Moroney S.E.
      • Benner S.A.
      Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet.
      ,
      • Benner S.A.
      • Karalkar N.B.
      • Hoshika S.
      • Laos R.
      • Shaw R.W.
      • Matsuura M.
      • Fajardo D.
      • Moussatche P.
      Alternative Watson-Crick synthetic genetic systems.
      ) synthesized UBPs that exploit orthogonal patterns of hydrogen bonding. This led to multiple discoveries about the molecular changes that can (and cannot) be tolerated in replicating units, especially how the fidelity of replication can be influenced by protonation, deprotonation, and multiple tautomeric forms of the nucleobases (
      • Eberlein L.
      • Beierlein F.R.
      • Hommes N.R.V.
      • Radadiya A.
      • Heil J.
      • Benner S.A.
      • Clark T.
      • Kast S.M.
      • Richards N.G.J.
      Tautomeric equilibria of nucleobases in the Hachimoji expanded genetic alphabet.
      ). These efforts led to the preparation of “hachimoji” DNA (
      • Hoshika S.
      • Leal N.A.
      • Kim M.-J.
      • Kim M.-S.
      • Karalkar N.B.
      • Kim H.-J.
      • Bates A.M.
      • Watkins Jr., N.E.
      • SantaLucia H.A.
      • Meyer A.J.
      • DasGupta S.
      • Piccirilli J.A.
      • Ellington A.D.
      • SantaLucia Jr., J.
      • Georgiadis M.M.
      • et al.
      Hachimoji DNA and RNA: a genetic system with eight building blocks.
      ), an artificially expanded genetic system (AEGIS) in which A, G, T, and C are augmented by two unnatural pyrimidine analogs, pseudocytidine (or isocytidine) (S) and 6-amino-3-(2′-deoxyribo-furanosyl)-5-nitro-1H-pyridin-2-one (Z), and their size and hydrogen bond complementary partners, the purine analogs, isoguanine (B), and 2-amino-8-(1-β-d-2′-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-[8H]-4-one- (P) (
      • Eberlein L.
      • Beierlein F.R.
      • Hommes N.R.V.
      • Radadiya A.
      • Heil J.
      • Benner S.A.
      • Clark T.
      • Kast S.M.
      • Richards N.G.J.
      Tautomeric equilibria of nucleobases in the Hachimoji expanded genetic alphabet.
      ,
      • Hoshika S.
      • Leal N.A.
      • Kim M.-J.
      • Kim M.-S.
      • Karalkar N.B.
      • Kim H.-J.
      • Bates A.M.
      • Watkins Jr., N.E.
      • SantaLucia H.A.
      • Meyer A.J.
      • DasGupta S.
      • Piccirilli J.A.
      • Ellington A.D.
      • SantaLucia Jr., J.
      • Georgiadis M.M.
      • et al.
      Hachimoji DNA and RNA: a genetic system with eight building blocks.
      ) (Fig. 1). The additional P:Z and B:S base pairs form three interbase hydrogen bonds, as does the natural G:C. Work in this area has also been driven by commercial factors; the use of AEGIS UBPs as components of diagnostic tests has generated lifetime sales in excess of $1 billion (
      • Glushakova L.G.
      • Alto B.W.
      • Kim M.-S.
      • Hutter D.
      • Bradley A.
      • Bradley K.M.
      • Burkett-Cadena N.D.
      • Benner S.A.
      Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya and dengue viruses in mosquitoes.
      ).
      Future disruptive technologies exploiting these and other expanded genetic alphabets will almost certainly require engineered DNA polymerase variants having expanded ranges of biophysical and catalytic properties. Unfortunately, and despite extensive research into the structure and mechanism of DNA polymerases from a wide range of organisms (
      • Raper A.T.
      • Reed A.J.
      • Suo Z.
      Kinetic mechanism of DNA polymerases: contributions of conformational dynamics and a third metal ion.
      ,
      • Wu W.-J.
      • Yang W.
      • Tsai M.-D.
      How DNA polymerases catalyse replication and repair with contrasting fidelity.
      ,
      • Raia P.
      • Delarue M.
      • Sauguet L.
      An updated structural classification of replicative DNA polymerases.
      ,
      • Steitz T.A.
      DNA polymerases: structural diversity and common mechanisms.
      ,
      • Johnson K.A.
      The kinetic and chemical mechanism of high-fidelity DNA polymerases.
      ,
      • Kiefer J.R.
      • Mao C.
      • Braman J.C.
      • Beese L.S.
      Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal.
      ,
      • Kropp H.M.
      • Dürr S.L.
      • Peter C.
      • Diederichs K.
      • Marx A.
      Snapshots of a modified nucleotide moving through the confines of a DNA polymerase.
      ), rationally predicting how specific amino acid replacements in these enzymes will impact the incorporation of any particular kind of UBP into duplex DNA remains almost impossible. This reflects a lack of fundamental understanding about how multiple factors act synergistically to control UBP incorporation efficiency and fidelity (
      • Raper A.T.
      • Reed A.J.
      • Suo Z.
      Kinetic mechanism of DNA polymerases: contributions of conformational dynamics and a third metal ion.
      ,
      • Joyce C.M.
      • Benkovic S.J.
      DNA polymerase fidelity: kinetics, structure, and checkpoints.
      ,
      • Kunkel T.A.
      • Bebenek K.
      DNA replication fidelity.
      ). These factors include nucleobase pair complementarity, nucleobase tautomerism, variations in hy-drogen bond free energies, and the conformational dynamics of the polymerase itself as incorporation proceeds through the catalytic cycle. Directed evolution methods (
      • Arnold F.H.
      Innovation by evolution: bringing new chemistry to life.
      ) are therefore viewed as the best approach to identifying DNA polymerase variants that can incorporate UBPs with high efficiency and fidelity (
      • Houlihan G.
      • Arangundy-Franklin S.
      • Holliger P.
      Exploring the chemistry of genetic information storage and propagation through polymerase engineering.
      ,
      • Chen T.
      • Romesberg F.E.
      Directed polymerase evolution.
      ,
      • Laos R.
      • Thomson J.M.
      • Benner S.A.
      DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides.
      ).
      These facts make it timely to review how X-ray crystallography, computational modeling, and directed evolution strategies can be combined to obtain DNA polymerases capable of replicating artificial DNA containing UBPs. Given the recent publication of a comprehensive discussion about the replication of UBP-containing DNA by unengineered WT polymerases (
      • Betz K.
      • Marx A.
      The structural basis for processing of unnatural base pairs by DNA polymerases.
      ), we draw on work from our laboratories, the only studies (to our knowledge) that employ a combined experimental and computational approach.
      Generally speaking, enzymes that replicate DNA must manage three contradictory demands as they catalyze their critical reactions. First, this class of polymerases must be highly specific, making not much more than one mistake every billion turnovers (
      • Fowler R.G.
      • Degnen G.E.
      • Cox E.C.
      Mutational specificity of a conditional Escherichia coli mutator, mutD5.
      ). Mistakes mean somatic mutations potentially leading to cancer, germ line mutations that create inherited genetic disease, and, ultimately, an error catastrophe that leads to the death of the organism. Typical replicative polymerases achieve specificity at both the elongation step and a proofreading step, the latter in the form of 3′–5′ exonuclease domains (see below). Other replicative DNA polymerases, such as reverse transcriptases (
      • Yu H.
      • Goodman M.F.
      Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates.
      ), lack proofreading and are therefore more error-prone. DNA polymerases that function primarily in DNA repair, including those important for genome stability and trans-lesion synthesis, also often have lower fidelity (
      • Brambati A.
      • Barry R.M.
      • Sfeir A.
      DNA polymerase theta (Polθ)—an error-prone polymerase necessary for genome stability.
      ,
      • Du H.
      • Wang P.
      • Wu J.
      • He X.
      • Wang Y.
      The roles of polymerases ν and θ in replicative bypass of O6- and N2-alkyl-2′-deoxyguanosine lesions in human cells.
      ).
      Second, DNA polymerases must accept four different substrates: (i) template dG and dCTP, (ii) template dC and dGTP, (iii) template dA and dTTP, and (iv) template dT and dATP. Most other enzymes accept only one.
      Finally, DNA polymerases must work rapidly. An E. coli cell replicates its entire genome by copying 4000 nucleotides each second. Even though many replication forks are used, this rate is as fast as that of many enzymes using only one substrate in pathways able to tolerate mistakes.
      Managing these demands is especially difficult because the four substrates have few molecular features in common to present to the polymerase (Fig. 1). For example, the four nucleobases present different functionalities to the major groove, a methyl group for T, a hydrogen for C, two hydrogen bond acceptors for G, and one donor and one acceptor for A.
      One feature that all four substrates have in common is their relative size, leading to the rule that large purines must pair with small pyrimidines. Further, the functional groups located on the sides of all four bases in the minor groove of the DNA duplex share a common feature: electron density presented by the exocyclic C=O groups of T and C and the N-3 nitrogen in the purine rings of A and G. Thus, the “minor groove scanning hypothesis” proposes that polymerases donate hydrogen bonds to this electron density in the template, primer, and incoming triphosphate as a way of enforcing Watson–Crick geometry, and hence fidelity, on base pair recognition (
      • Hendrickson C.
      • Devine K.
      • Benner S.A.
      Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2′-deoxyadenosine.
      ).
      These hydrogen bond contacts and size constraints are well-represented in over two dozen crystal structures solved to date (
      • Raia P.
      • Delarue M.
      • Sauguet L.
      An updated structural classification of replicative DNA polymerases.
      ,
      • Betz K.
      • Marx A.
      The structural basis for processing of unnatural base pairs by DNA polymerases.
      ). The electronic constraint in the minor groove also appears to be important for nonstandard base pairing, even pairing that, as in the Romesberg pair (Fig. 1), involves no interbase hydrogen bonding (
      • Powner M.W.
      • Gerland B.
      • Sutherland J.D.
      Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions.
      ). Moreover, the easiest AEGIS UBP to incorporate into DNA by polymerase-catalyzed reactions has been Z:P (Fig. 1), which is the only pair of those with shuffled hydrogen-bonding patterns for which both components present electron density to the minor groove (
      • Benner S.A.
      Understanding nucleic acids using synthetic chemistry.
      ).
      Although the coupling reaction catalyzed by DNA polymerases seems superficially straightforward, this simplicity hides many details that remain controversial (
      • Raper A.T.
      • Reed A.J.
      • Suo Z.
      Kinetic mechanism of DNA polymerases: contributions of conformational dynamics and a third metal ion.
      ,
      • Johnson K.A.
      The kinetic and chemical mechanism of high-fidelity DNA polymerases.
      ,
      • Joyce C.M.
      • Steitz T.A.
      Function and structure relationships in DNA polymerases.
      ,
      • Kuchta R.D.
      • Benkovic P.
      • Benkovic S.J.
      Kinetic mechanism whereby DNA polymerase-I (Klenow) replicates DNA with high fidelity.
      ,
      • Steitz T.A.
      DNA- and RNA-dependent DNA polymerases.
      ). DNA synthesis proceeds in the 5′ to 3′ direction in a primer-dependent reaction requiring Mg2+ as a cofactor. In the first step, the enzyme forms an initial binary complex with a DNA duplex composed of a template strand and a shorter, complementary primer strand possessing a 3′-hydroxyl group. dNTPs then bind to the binary complex to give a ternary (or “pre-incorporation”) complex (Fig. 2). Correctly matched dNTPs reside longer in the active site, allowing the enzyme to adopt a “closed” conformation in which the 3′-hydroxyl group in the primer can attack the α-phosphate of the bound dNTP (Fig. 2A). This reaction requires the presence of two (
      • Tsai Y.C.
      • Johnson K.A.
      A new paradigm for DNA polymerase specificity.
      ,
      • Yoon H.
      • Warshel A.
      Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis.
      ), or possibly three (
      • Raper A.T.
      • Reed A.J.
      • Suo Z.
      Kinetic mechanism of DNA polymerases: contributions of conformational dynamics and a third metal ion.
      ,
      • Steitz T.A.
      A mechanism for all polymerases.
      ,
      • Gao Y.
      • Yang W.
      Capture of a third Mg2+ is essential for catalyzing DNA synthesis.
      ), Mg2+ ions within the active site of human polymerase β (Fig. 2C) or Klentaq (Fig. 2D). Subsequent release of PPi generates a binary (or “post-incorporation”) complex in which the primer has been elongated by one nucleobase, which then serves as the substrate for the next templated addition of dNTP. These steps are repeated until no unpaired nucleobases remain in the template strand.
      Figure thumbnail gr2
      Figure 2A, kinetic scheme for the incorporation of matched dNTPs by T7 DNA polymerase (
      • Kunkel T.A.
      • Bebenek K.
      DNA replication fidelity.
      ,
      • Tsai Y.C.
      • Johnson K.A.
      A new paradigm for DNA polymerase specificity.
      ). When the incoming dNTP is correct, the enzyme undergoes a rapid conformational change from the open to closed form of the pre-incorporation complex. B, kinetic scheme for the incorporation of mis-matched dNTPs by T7 DNA polymerase (
      • Kunkel T.A.
      • Bebenek K.
      DNA replication fidelity.
      ,
      • Tsai Y.C.
      • Johnson K.A.
      A new paradigm for DNA polymerase specificity.
      ). When the incoming dNTP is incorrect, the rate constant for the chemical step becomes smaller than that for the conformational change back to the open form of the pre-incorporation complex. C, chemical mechanism of primer elongation for the 2-Mg (
      • Steitz T.A.
      A mechanism for all polymerases.
      ) and 3-Mg models (
      • Gao Y.
      • Yang W.
      Capture of a third Mg2+ is essential for catalyzing DNA synthesis.
      ). The location of the third Mg2+ ion together with its water ligands is shown in red. Residue numbering is for human DNA polymerase β. D, chemical mechanism of primer elongation in the active site of T. aquaticus DNA polymerase, which lacks one of the carboxylate ligands present in human DNA polymerase β.
      Replicative DNA polymerases, especially those for which DNA binding is the rate-limiting step, exhibit a property referred to as processivity (
      • Bambara R.A.
      • Uyemura D.
      • Choi T.
      On the processive mechanism of DNA polymerase I: quantitative assessment of processivity.
      ). Processivity is a measure of the number of dNTPs incorporated into the growing strand before the template-primer duplex dissociates from the enzyme (
      • Korangy F.
      • Julin D.A.
      A mutation in the consensus ATP-binding sequence of the RecD subunit reduces the processivity of the RecBCD enzyme from Escherichia coli.
      ). Processivity, of course, permits these DNA polymerases to achieve high rates of nucleotide incorporation. This process requires translocation of the enzyme along the template-primer or the template-primer along the enzyme (depending on your point of view) during polymerization. We discuss the conformational changes that occur during dNTP incorporation below.
      When mismatched dNTPs are present in the ternary complex, kinetic evidence shows that the active site remains in an “open” conformation so that dissociation of the incoming nucleotide can take place prior to formation of the new P–O bond (
      • Joyce C.M.
      • Benkovic S.J.
      DNA polymerase fidelity: kinetics, structure, and checkpoints.
      ,
      • Kunkel T.A.
      • Bebenek K.
      DNA replication fidelity.
      ) (Fig. 2B). Alternatively, should a mismatched dNTP be incorporated, most replicative polymerases include a 3′–5′ exonuclease domain that can remove the mismatch. In this case, the 3′-hydroxyl end of the primer shifts from the polymerase to the exonuclease active site for removal of the mismatched nucleotide (
      • Donlin M.J.
      • Patel S.S.
      • Johnson K.A.
      Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction.
      ,
      • Fidalgo da Silva E.
      • Reha-Krantz L.J.
      DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase.
      ). This proofreading step is essential for ensuring fidelity during replication by DNA polymerases, which typically make errors every 104 to 105 nucleobases. The 3′–5′ exonucleolytic activity improves this error rate by 2–3 orders of magnitude (
      • Bebenek A.
      • Ziuzia-Graczyk I.
      Fidelity of DNA replication—a matter of proof-reading.
      ).
      A variety of assays exist to assess the kinetics of nucleobase incorporation and processivity. These have been extensively reviewed (
      • Johnson K.A.
      The kinetic and chemical mechanism of high-fidelity DNA polymerases.
      ,
      • Berdis A.J.
      Mechanisms of DNA polymerases.
      ), but here we give two examples used in our work to determine either incorporation efficiency (
      • Yang Z.
      • Chen F.
      • Alvarado J.B.
      • Benner S.A.
      Amplification, mutation, and sequencing of a six-letter synthetic genetic system.
      ) or fidelity (
      • Chen F.
      • Yang Z.Y.
      • Yang M.C.
      • Alvarado J.B.
      • Wang G.G.
      • Benner S.A.
      Recognition of an expanded genetic alphabet by type II restriction endonucleases and their application to analyze polymerase fidelity.
      ) when DNA polymerases and their variants are challenged with AEGIS UBPs (Fig. 3). For example, incorporation efficiency can be measured by nested PCR using primers tagged at the 5′-end with multiple consecutive UBPs. In this assay, amplicons are formed in the reaction mixture only if the polymerase is capable of replicating the UBPs (Fig. 3A).
      Figure thumbnail gr3
      Figure 3A, nested PCR-based incorporation assay for DNA containing Z:P nucleobase pairs using a target DNA sequence (cyan-red-green) composed of only WC base pairs. Replication is initiated by chimeric primers that can bind to the original gene but have UBPs at the 5′-end. These chimeric primers are present at low concentration (Step 1). After consumption of the chimeric primers (Step 2), amplification is continued by external 5′-PPPP-OH primers (containing only UBPs), which are present at high concentration. As a result, full-length amplicons are produced only if the DNA polymerase can efficiently incorporate UBPs (based on of Ref.
      • Laos R.
      • Shaw R.
      • Leal N.A.
      • Gaucher E.
      • Benner S.
      Directed evolution of polymerases to accept nucleotides with non-standard hydrogen-bond patterns.
      ). B, PCR-based replication fidelity assay for DNA containing Z:P nucleobase pairs. If the UBP is lost to a T:A base pair, then a Dra restriction site (red) is generated in the amplicons. Similarly, if the UBP is lost to a C:G base pair, then an Alu restriction site (blue) is produced. C, gel showing misincorporation rates in the PCR amplification of template DNA in the presence of dZTP and/or dPTP components using WT Klentaq and WT Phusion DNA polymerases at different pH values (
      • Chen F.
      • Yang Z.Y.
      • Yang M.C.
      • Alvarado J.B.
      • Wang G.G.
      • Benner S.A.
      Recognition of an expanded genetic alphabet by type II restriction endonucleases and their application to analyze polymerase fidelity.
      ). Four parallel PCRs were performed followed by digestion with PspOMI for 16 h. The ratio between the amount of full-length product (FLP) and all oligonucleotides gives the misincorporation rates shown in the figure. Lane 1, negative control PCR amplification of template DNA in the presence of WC dNTPs (200 mm each), followed by digestion with PspOMI. Lane 2, five-letter PCR amplification of template DNA in the presence of WC dNTPs (200 mm each) and dZTP (25 mm), followed by digestion with PspOMI. Lane 3, five-letter PCR amplification of template DNA in the presence of WC dNTPs (200 mm each) and dPTP (25 mm), followed by digestion with PspOMI. Lane 4, six-letter PCR amplification of template DNA in the presence of WC dNTPs (200 mm each), dZTP (25 mm), and dPTP (25 mm), followed by digestion with PspOMI. Taken from Ref.
      • Chen F.
      • Yang Z.Y.
      • Yang M.C.
      • Alvarado J.B.
      • Wang G.G.
      • Benner S.A.
      Recognition of an expanded genetic alphabet by type II restriction endonucleases and their application to analyze polymerase fidelity.
      and used with permission.
      Assaying fidelity requires the DNA polymerase to replicate a sequence in which UBPs are present (
      • Chen F.
      • Yang Z.Y.
      • Yang M.C.
      • Alvarado J.B.
      • Wang G.G.
      • Benner S.A.
      Recognition of an expanded genetic alphabet by type II restriction endonucleases and their application to analyze polymerase fidelity.
      ) (Fig. 3B). In the event that the UBP is lost during PCR cycles, two possible unique restriction sites are created in the amplicons, depending on whether the UBP is replaced by T:A or C:G. Incubating the PCR products with two restriction endonucleases therefore gives cleaved products for the amplicons from which the UBP has been lost in both possible transition mutations (Fig. 3C). This assay thereby reports on the extent to which polymerase fidelity deviates from 100%. In addition, performing the PCR with different concentrations of the UBP components permits a quantitative assessment of fidelity.

      Obtaining DNA polymerases engineered for altered substrate specificity

      Some, albeit limited, success has been reported in modifying the substrate preferences of DNA polymerases by structure-guided mutagenesis studies (
      • Astatke M.
      • Ng K.
      • Grindley N.D.
      • Joyce C.M.
      A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides.
      ,
      • Gardner A.F.
      • Jack W.E.
      Acyclic and dideoxy terminator preference denote divergent sugar recognition by archaeon and Taq DNA polymerases.
      ). The complexity of the conformational changes undergone by the polymerase during each catalytic cycle seems to require that variants contain multiple residue replacements. Most successful efforts to obtain DNA polymerases with altered catalytic and/or biophysical properties have therefore selected suitable variants from large libraries (
      • Houlihan G.
      • Arangundy-Franklin S.
      • Holliger P.
      Exploring the chemistry of genetic information storage and propagation through polymerase engineering.
      ,
      • Coulther T.A.
      • Stern H.R.
      • Beuning P.J.
      Engineering polymerases for new functions.
      ,
      • Aschenbrenner J.
      • Werner S.
      • Marchand V.
      • Adam M.
      • Motorin Y.
      • Helm M.
      • Marx A.
      Engineering of a DNA polymerase for direct m6A sequencing.
      ,
      • Chen T.
      • Hongdilokkul N.
      • Liu Z.
      • Adhikary R.
      • Tsuen S.S.
      • Romesberg F.E.
      Evolution of thermophilic DNA polymerases for the recognition and amplification of C2′-modified DNA.
      ,
      • Schultz H.J.
      • Gochi A.M.
      • Chia H.E.
      • Ogonowsky A.L.
      • Chiang S.
      • Filipovic N.
      • Weiden A.G.
      • Hadley E.E.
      • Gabriel S.E.
      • Leconte A.M.
      Taq DNA polymerase mutants and 2′-modified sugar recognition.
      ,
      • Ellefson J.W.
      • Gollihar J.
      • Shroff R.
      • Shivram H.
      • Iyer V.R.
      • Ellington A.D.
      Synthetic evolutionary origin of a proofreading reverse transcriptase.
      ). Examples include several Taq polymerase variants evolved for specific functions (Table 1). The fact that several of the amino acid substitutions are found in more than one of these Taq variants may ultimately inform rationale design of variants with new functional properties in the future. To date, however, only one of these published variants (ZP Klentaq) has been subjected to structural and dynamic characterizations to determine the impact of each of the various substitutions on the properties of the variant polymerase (see below).
      Table 1Examples of evolved Taq DNA polymerase variants
      Taq variantSubstrate/PropertyAmino acid substitutionsReference
      SFM4-3C2′ modificationI614E/E615G/N583S/D655N/E681K/E742Q/M747R
      • Chen T.
      • Hongdilokkul N.
      • Liu Z.
      • Adhikary R.
      • Tsuen S.S.
      • Romesberg F.E.
      Evolution of thermophilic DNA polymerases for the recognition and amplification of C2′-modified DNA.
      AA40RNA, DNAE602V/A608V/I614M/E615G
      • Ong J.L.
      • Loakes D.
      • Jaroslawski S.
      • Too K.
      • Holliger P.
      Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.
      M13′ mismatchesG84A/D144G/K314R/E520G/F598L/A608V/E742G
      • Ghadessy F.J.
      • Ramsay N.
      • Boudsocq F.
      • Loakes D.
      • Brown A.
      • Iwai S.
      • Vaisman A.
      • Woodgate R.
      • Holliger P.
      Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution.
      M43′ mismatchesD58G, R74P, A109T, L245R, R343G, G370D, E520G, N583S, E694K, A743P
      • Ghadessy F.J.
      • Ramsay N.
      • Boudsocq F.
      • Loakes D.
      • Brown A.
      • Iwai S.
      • Vaisman A.
      • Woodgate R.
      • Holliger P.
      Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution.
      5D4Hydrophobic basesV62I, Y78H, T88S, P114Q, P264S, E303V, G389V, E424G, E432G, E602G, A608V, I614M, M761T, M775T
      • Loakes D.
      • Gallego J.
      • Pinheiro V.B.
      • Kool E.T.
      • Holliger P.
      Evolving a polymerase for hydrophobic base analogues.
      H15ThermostabilityK225E/E388V/K540R/D578G/N583S/M747R
      • Ghadessy F.J.
      • Ong J.L.
      • Holliger P.
      Directed evolution of polymerase function by compartmentalized self-replication.
      RT-KTQm6-A sequencingL459M/S515R/I638F/M747K
      • Aschenbrenner J.
      • Werner S.
      • Marchand V.
      • Adam M.
      • Motorin Y.
      • Helm M.
      • Marx A.
      Engineering of a DNA polymerase for direct m6A sequencing.
      ZPZ:P substratesM444V/P527A/E551E/E832V
      • Laos R.
      • Shaw R.
      • Leal N.A.
      • Gaucher E.
      • Benner S.
      Directed evolution of polymerases to accept nucleotides with non-standard hydrogen-bond patterns.
      Of the plethora of directed evolution strategies, compartmentalized self-replication (CSR) (
      • Ghadessy F.J.
      • Ong J.L.
      • Holliger P.
      Directed evolution of polymerase function by compartmentalized self-replication.
      ,
      • Tawfik D.
      • Griffiths A.D.
      Man-made cell-like compartments for molecular evolution.
      ) or one of its modifications (
      • Houlihan G.
      • Arangundy-Franklin S.
      • Holliger P.
      Exploring the chemistry of genetic information storage and propagation through polymerase engineering.
      ,
      • Coulther T.A.
      • Stern H.R.
      • Beuning P.J.
      Engineering polymerases for new functions.
      ) is often used to obtain engineered DNA polymerases. In CSR, water droplets containing appropriate primers and dNTPs are formed in a water/oil emulsion. Droplets that also contain a single E. coli cell carrying a plasmid encoding a single variant of a thermophilic DNA polymerase, such as Klentaq, can then be used to select enzymes with desired properties. In our work (
      • Laos R.
      • Shaw R.
      • Leal N.A.
      • Gaucher E.
      • Benner S.
      Directed evolution of polymerases to accept nucleotides with non-standard hydrogen-bond patterns.
      ), we employ the nested PCR strategy described above (Fig. 3A) to identify DNA polymerase variants that can replicate UBPs orthogonal to WC nucleobases, such as Z and P, because only these will replicate copies of their gene in the plasmid (Fig. 4). Those variants that cannot incorporate UBPs efficiently will not replicate their genes. Thus, genes encoding DNA polymerases with the desired substrate specificity become represented more in the mixture of DNA amplicons obtained when the emulsion is broken at the end of several rounds of PCR (Fig. 4). The resulting DNA amplicons are inserted into a new set of plasmids that are used to transform E. coli for use in a subsequent selection round. After a defined number of rounds, genes encoding DNA polymerase variants with the ability to incorporate UBPs become highly enriched in the library. The set of enriched genes can be sequenced to identify the molecular changes that permit the polymerase variant to replicate the UBP. Standard methods can then be used to express and purify individual variants prior to any detailed evaluation of their fidelity and incorporation efficiencies (
      • Yang Z.
      • Chen F.
      • Alvarado J.B.
      • Benner S.A.
      Amplification, mutation, and sequencing of a six-letter synthetic genetic system.
      ,
      • Chen F.
      • Yang Z.Y.
      • Yang M.C.
      • Alvarado J.B.
      • Wang G.G.
      • Benner S.A.
      Recognition of an expanded genetic alphabet by type II restriction endonucleases and their application to analyze polymerase fidelity.
      ).
      Figure thumbnail gr4
      Figure 4A, overview of a modified CSR strategy for identifying DNA polymerase variants capable of replicating AEGIS UBPs from a library. Each E. coli cell carries a thermophilic DNA polymerase variant (Step 1) and is emulsified with dNTPs and nested primers within a single water droplet. Heating lyses the cells and inactivates endogenous mesophilic polymerases (Step 2). PCR amplification is then performed (Step 3). Polymerases that can replicate UBPs (red) amplify their respective genes, enriching the pool of variant genes in the pool; those that cannot replicate UBPs fail to do so, so their genes are lost (Step 4). The amplified genes encoding polymerase variants that can replicate UBPs are extracted after breaking the emulsion and used to create a new library of plasmids (Step 5) that can be subjected to a further selection round (Step 6). After an appropriate number of selection rounds, the pool of enriched genes can be sequenced (Step 7) and used to express polymerase variants that can be characterized using standard in vitro methods. Based on of Ref.
      • Laos R.
      • Shaw R.
      • Leal N.A.
      • Gaucher E.
      • Benner S.
      Directed evolution of polymerases to accept nucleotides with non-standard hydrogen-bond patterns.
      . B, In CST, each E. coli cell carries a thermophilic DNA polymerase variant encoded on a plasmid (Step 1), which also contains a sequence containing one or more UBPs complementary to a single biotin-linked (“tagged”) primer and is emulsified with dNTPs and the tagged primer within a single water droplet. Heating lyses the cells and inactivates endogenous mesophilic polymerases (Step 2). Subsequent DNA replication (Step 3) by the thermophilic DNA polymerase variant results in a “biotin-tagged” form of the plasmid containing the gene (red) encoding the polymerase variant that can replicate the UBP(s). Polymerases that cannot replicate the UBP (blue) will only give weakly stable primer-plasmid complexes. After breaking the emulsion, tagged plasmids are captured onto a bead (Step 4) in proportion to the stability of the extended primer-plasmid complex (Step 5). The recovered plasmid DNA is then amplified and used in a new round of selection (Step 6) or screened (Step 7). Based on Fig. S1 of Ref.
      • Pinheiro V.B.
      • Taylor A.I.
      • Cozens C.
      • Abramov M.
      • Renders M.
      • Zhang S.
      • Chaput J.C.
      • Wengel J.
      • Peak-Chew S.Y.
      • McLaughlin S.H.
      • Herdewijn P.
      • Holliger P.
      Synthetic genetic polymers capable of heredity and evolution.
      ).
      The original CSR strategy, however, requires the replication of the entire polymerase gene, which contains more than 2000 nucleobases. Thus, only the most active variants are recovered. Many technical aspects, including the appropriate choice of oil or surfactant containing the water droplets (
      • Lu W.-C.
      • Ellington A.D.
      In vitro selection of proteins via emulsion compartments.
      ,
      • Davidson E.A.
      • Diugosz P.J.
      • Levy M.
      • Ellington A.D.
      Directed evolution of proteins in vitro using compartmentalization in emulsions.
      ), must be optimized if reproducible results are to be obtained (
      • Miller O.J.
      • Bernath K.
      • Agresti J.J.
      • Amitai G.
      • Kelly B.T.
      • Mastrobattista E.
      • Taly V.
      • Magdassi S.
      • Tawfik D.S.
      • Griffiths A.D.
      Directed evolution by in vitro compartmentalization.
      ).
      Improved variations of this selection method have therefore been reported by Philip Holliger and co-workers (
      • Pinheiro V.B.
      • Taylor A.I.
      • Cozens C.
      • Abramov M.
      • Renders M.
      • Zhang S.
      • Chaput J.C.
      • Wengel J.
      • Peak-Chew S.Y.
      • McLaughlin S.H.
      • Herdewijn P.
      • Holliger P.
      Synthetic genetic polymers capable of heredity and evolution.
      ,
      • Pinheiro V.B.
      • Arangundy-Franklin S.
      • Holliger P.
      Compartmentalized self-tagging for in vitro-directed evolution of XNA polymerases.
      ), including compartmentalized self-tagging (CST) (Fig. 4B). In CST, the positive feedback loop depends on the polymerase “tagging” a plasmid containing its encoding gene by extension of a biotinylated oligonucleotide. As a result, plasmids containing genes that encode DNA polymerases with altered substrate selectivity can be enriched by selective capture onto an affinity column. Clearly, the UBP(s) must be located within a segment of plasmid DNA that is replicated with high efficiency and fidelity by the DNA polymerase variant. In addition, the sensitivity of the assay is higher because CST does not require multiple self-replications of the complete gene encoding the polymerase variant.

      Insights from structural studies of WT and evolved Klentaq DNA polymerase complexes

      In contrast to some other critical cellular enzymes and machines, DNA polymerases have been reinvented throughout evolution (
      • Raia P.
      • Delarue M.
      • Sauguet L.
      An updated structural classification of replicative DNA polymerases.
      ). As a consequence, several distinct structural classes of polymerases provide multiple opportunities to identify an enzyme that will efficiently and faithfully replicate an unnatural base pair. Replicative DNA polymerases have been classified as families A–D, with D being the most recently identified DNA polymerase family from Archaea (
      • Raia P.
      • Delarue M.
      • Sauguet L.
      An updated structural classification of replicative DNA polymerases.
      ).
      To date, naturally occurring family A and B DNA polymerases have been used to successfully replicate single UBPs. Family A polymerases are found primarily in bacteria; their peptide fold is described as including fingers, palm, and thumb domains associated with polymerase activity (Fig. 5A). They also have 3′–5′ exonuclease and 5′–3′ exonuclease domains, the second removing the RNA primers required for lagging strand synthesis (
      • Raia P.
      • Delarue M.
      • Sauguet L.
      An updated structural classification of replicative DNA polymerases.
      ). In Klentaq, the Thermus aquaticus DNA polymerase (
      • Kim Y.
      • Eom S.H.
      • Wang J.
      • Lee D.-S.
      • Suh S.W.
      • Steitz T.A.
      Crystal structure of Thermus aquaticus DNA polymerase.
      ), corresponding to the Klenow or large fragment of the enzyme (
      • Ollis D.L.
      • Brick P.
      • Hamlin R.
      • Xuong N.G.
      • Steitz T.A.
      Structure of large fragment of Escherichia coli DNA polymerase-I complexed with dTMP.
      ), the N-terminal 5′–3′ exonuclease domain is absent, and the 3′–5′ exonuclease domain lacks activity. In contrast, family B DNA polymerases, found in all archaea, include a 3′–5′ exonuclease domain and a polymerase domain, which is related to that in family A polymerases (
      • Raia P.
      • Delarue M.
      • Sauguet L.
      An updated structural classification of replicative DNA polymerases.
      ).
      Figure thumbnail gr5
      Figure 5Structural properties of Klentaq DNA polymerase. A, the open form of ZP Klentaq (from PDB entry 5W6Q), shown in a cartoon rendering, includes fingers (blue), palm (green), thumb (yellow), and 3′–5′ exonuclease (red) domains. This architecture is conserved in all family A polymerases. Variant amino acid substitutions are shown as sphere models (orange, carbon; blue, nitrogen; red, oxygen), M444V, P527A, and D551E. (The fourth amino acid substitution, E832V, is disordered in the structures). α-Helices G, H, and I contribute to dynamic motion in ZP Klentaq. B, a similar rendering of ZP Klentaq in the binary, post-incorporation complex (PDB entry 5W6Q) is shown with template-primer, including P:Z in the active site (magenta cartoon rendering). C, the pre-incorporation complex of ZP Klentaq (PDB entry 5W6K) is shown superimposed on the post-incorporation complex from B. The black arrow indicates the most significant conformational change associated with the fingers domain (light blue for the initial and post-incorporation and blue for the pre-incorporation complex). The O helix closes down, forming the active site in the ternary complex with bound dZTP (magenta stick rendering). The thumb domains show a slight shift in position (light orange for post-incorporation and yellow for pre-incorporation complex). The palm and exonuclease domains show no significant differences in overall position in the two complexes. The substrate nucleic acid is shown as a cartoon rendering (pink in the post-incorporation and magenta in the post-incorporation complex). Close-ups of hydrogen-bonding interactions in the active site for the WT Klentaq bound to ddCTP-dG and dZTP-dP are shown in D and E, respectively. In each case, the dNTP is positioned through a water-mediated hydrogen-bonding interaction involving O2 (minor groove edge of the nucleobase) with Gln-754. Similarly, the penultimate primer nucleobase presents either N3 or O2 for hydrogen-bonding to Arg-573 (again a minor groove interaction). In the case of the ZP Klentaq ternary complex (E), Arg-660 hydrogen-bonds to the NO2 group present on the major groove edge of Z. F, the active site including template-primer and dZTP (magenta stick rendering), Mg2+ ions (orange spheres), and coordinating residues Asp-610 and Asp-785 (green stick rendering) are shown with a ribbon rendering of the enzyme, palm in green and fingers in blue.
      Hydrophobic nonhydrogen-bonding pairs developed by Romesberg (NAM:TPT3) (
      • Kool E.T.
      Replacing the nucleobases in DNA with designer molecules.
      ,
      • Feldman A.W.
      • Romesberg F.E.
      Expansion of the genetic alphabet: a chemist's approach to synthetic biology.
      ) and Hirao (Ds:Px) (
      • Malyshev D.A.
      • Romesberg F.E.
      The expanded genetic alphabet.
      ,
      • Piccirilli J.A.
      • Krauch T.
      • Moroney S.E.
      • Benner S.A.
      Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet.
      ) have been replicated with selectivities exceeding 99% using DeepVent DNA polymerase (family B) (
      • Malyshev D.A.
      • Dhami K.
      • Lavergne T.
      • Chen T.
      • Dai N.
      • Foster J.M.
      • Corrèa Jr., I.R.
      • Romesberg F.E.
      A semi-synthetic organism with an expanded genetic alphabet.
      ) or a combination of DeepVent DNA polymerase and Taq DNA polymerase (family A) (
      • Yamashige R.
      • Kimoto M.
      • Takezawa Y.
      • Sato A.
      • Mitsui T.
      • Yokoyama S.
      • Hirao I.
      Highly specific unnatural base pair systems as a third base pair for PCR amplification.
      ), respectively. It has proven much more difficult, however, to replicate templates containing consecutive hydrophobic nucleobases. The problem appears to be caused, in part, by disruptions to the DNA double helix resulting from the presence of hydrophobic UBPs (
      • Negi I.
      • Kathuria P.
      • Sharma P.
      • Wetmore S.D.
      How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulations.
      ). For example, the dNaM:d5SICS pair adopts an intercalative stacking mode in duplex DNA, as seen in both NMR structures and binary template-primer Klentaq complexes (
      • Malyshev D.A.
      • Pfaff D.A.
      • Ippoliti S.I.
      • Hwang G.T.
      • Dwyer T.J.
      • Romesberg F.E.
      Solution structure, mechanism of replication, and optimization of an unnatural base pair.
      ,
      • Betz K.
      • Malyshev D.A.
      • Lavergne T.
      • Welte W.
      • Diederichs K.
      • Romesberg F.E.
      • Marx A.
      Structural insights into DNA replication without hydrogen bonds.
      ). At least 6 natural base pairs must separate Ds-Px pairs if replication is to be successful (
      • Kimoto M.
      • Kawai R.
      • Mitsui T.
      • Yokoyama S.
      • Hirao I.
      An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules.
      ); as yet there is no report of DNA containing consecutive NaM:PTP3 (or structurally related hydrophobic UBPs) being replicated. Of course, the necessity of replicating more than one UBP, either consecutive or separted by standard WC nucleobases depends on the biotechnological application (
      • Fischer E.C.
      • Hashimoto K.
      • Zhang Y.
      • Feldman A.W.
      • Dien V.T.
      • Karadeema R.J.
      • Adhikary R.
      • Ledbetter M.P.
      • Krishnamurthy R.
      • Romesberg F.E.
      New codons for efficient production of unnatural proteins in a semisynthetic organism.
      ).
      A truly artificial genome would presumably include consecutive UBPs just as natural genomes include runs of sequential A:T or G:C pairs; cells possessing such a genome would require a DNA polymerase that can replicate through these regions of sequence. We have shown that up to 4 consecutive Z:P pairs can be incorporated by WT Klentaq polymerase (
      • Yang Z.
      • Chen F.
      • Alvarado J.B.
      • Benner S.A.
      Amplification, mutation, and sequencing of a six-letter synthetic genetic system.
      ); these UBP tetrads do not adversely impact the ability of duplex DNA to adopt A- and B-conformations (
      • Georgiadis M.M.
      • Singh I.
      • Kellett W.F.
      • Hoshika S.
      • Benner S.A.
      • Richards N.G.J.
      Structural basis for a six nucleotide genetic alphabet.
      ,
      • Molt Jr., R.W.
      • Georgiadis M.M.
      • Richards N.G.J.
      Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 µs molecular dynamics simulations.
      ). Unfortunately, the efficiency of Z:P incorporation by WT Klentaq is only 99.8% per theoretical PCR cycle (
      • Yang Z.
      • Chen F.
      • Alvarado J.B.
      • Benner S.A.
      Amplification, mutation, and sequencing of a six-letter synthetic genetic system.
      ), meaning that the UBP is easily lost during PCR-based applications. An engineered Klentaq variant (ZP Klentaq), exhibiting improved fidelity and Z:P incorporation efficiency was, however, obtained using CSR (Fig. 4A) (
      • Laos R.
      • Shaw R.
      • Leal N.A.
      • Gaucher E.
      • Benner S.
      Directed evolution of polymerases to accept nucleotides with non-standard hydrogen-bond patterns.
      ). Remarkably, ZP Klentaq contains only four amino acid substitutions, M444V, P527A, D551E, and E832V, all of which are located distal from the active site (Fig. 5A).
      In family A DNA polymerases, a large conformational change in the fingers domain occurs upon binding of the complementary dNTP, resulting in the formation of a closed complex that facilitates incorporation (Fig. 5). Of the structurally characterized DNA polymerases related to Klentaq at the sequence level (Table 2), only three have been captured in both pre- and post-incorporation complexes: ZP Klentaq (
      • Singh I.
      • Laos R.
      • Hoshika S.
      • Benner S.A.
      • Georgiadis M.M.
      Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide.
      ), WT Klentaq (
      • Betz K.
      • Malyshev D.A.
      • Lavergne T.
      • Welte W.
      • Diederichs K.
      • Dwyer T.J.
      • Ordoukhanian P.
      • Romesberg F.E.
      • Marx A.
      Klentaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry.
      ), and WT Geobacillus kaustophilus DNA polymerase (
      • Wang W.
      • Wu E.Y.
      • Hellinga H.W.
      • Beese L.S.
      Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides.
      ). Incorporation of nucleotides by the Geobacillus enzyme exhibits a fingers domain closure angle of 37°. In contrast to the Geobacillus enzyme, incorporation of natural dNTPs by WT Klentaq involves a much larger conformational change in which the fingers domain rotates by ∼59° (Fig. 5, B and C). In the closed conformation of the polymerase, correctly paired nucleotides are selected by hydrogen bonding and size complementarity. Despite retaining these features, Z:P pairs are more efficiently and faithfully incorporated by ZP Klentaq, in which the fingers close down by ∼64° (Table 3). Protein-nucleic acid interactions in both pre- and post-incorporations for ZP Klentaq are similar to those observed in the analogous WT complexes, suggesting that the evolved polymerase closely mimics the WT enzyme in this regard (Fig. 5, D and E).
      Table 2Structure-based alignments of Taq-related DNA polymerases
      PDBTypeSourceNo. of aligned Cα atomsRMSDSequence identity
      Å%
      5DKUApoPlasmodium418/5212.824.6
      4DQSBinaryG. kaustophilus486/5211.842.8
      1KFDApoE. coli429/5211.942.7
      2BPDBinaryGeobacilllus stearothermophilus488/5211.843.4
      4XVIBinaryHomo sapiens (Pol ν)439/5212.531.9
      6VDEApoMycobacterium smegmatis487/5212.140.0
      4 × 0QTernaryH. sapiens (Pol θ)443/5222.429.6
      Table 3Comparisons of binary and ternary complexes
      PDB file 1PDB file 2Fixed domainMoving domainRotation angleTranslationClosureBending residues
      degreesÅ%
      5W6K ZP tern5W6Q ZP Bin C299–637638–67163.6−1.788.8632–638
      672–829670–704
      3RTV3SZ2295–637638–67159.6−1.579.4626-638
      672–830669–696
      3KTQ4KTQ296–636637–67058.2−1.967.8626–637
      671–829670–686
      4DQQ*4DQS*300–679680–71636.6−0.786.6676–680
      717–874712–744
      The post-incorporation ZP Klentaq complex (PDB entry 5W6Q) (
      • Singh I.
      • Laos R.
      • Hoshika S.
      • Benner S.A.
      • Georgiadis M.M.
      Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide.
      ) also exhibits increased thumb domain motion (∼4°) relative to the analogous WT Klentaq complex (PDB entry 3SZ2) (
      • Betz K.
      • Malyshev D.A.
      • Lavergne T.
      • Welte W.
      • Diederichs K.
      • Dwyer T.J.
      • Ordoukhanian P.
      • Romesberg F.E.
      • Marx A.
      Klentaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry.
      ). Although Z:P pairs are readily accommodated in both B- and A-form DNA duplexes (
      • Georgiadis M.M.
      • Singh I.
      • Kellett W.F.
      • Hoshika S.
      • Benner S.A.
      • Richards N.G.J.
      Structural basis for a six nucleotide genetic alphabet.
      ,
      • Molt Jr., R.W.
      • Georgiadis M.M.
      • Richards N.G.J.
      Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 µs molecular dynamics simulations.
      ) and retain standard features (groove widths, base stacking parameters, etc.) consistent with these helical forms, modeling of a post-incorporation complex of P:Z bound to the WT Klentaq suggested that some structural alterations within the active site would be required to accommodate the UBP (
      • Singh I.
      • Laos R.
      • Hoshika S.
      • Benner S.A.
      • Georgiadis M.M.
      Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide.
      ). The two base pairs closest to the site of incorporation of the next dNTP exhibit A-form, which is significantly wider than B-form. This feature of the active site may allow WT Klentaq to accommodate a single hydrophobic UBP, which is wider than hydrogen-bonded pairs (
      • Betz K.
      • Malyshev D.A.
      • Lavergne T.
      • Welte W.
      • Diederichs K.
      • Dwyer T.J.
      • Ordoukhanian P.
      • Romesberg F.E.
      • Marx A.
      Klentaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry.
      ,
      • Betz K.
      • Kimoto M.
      • Diederichs K.
      • Hirao I.
      • Marx A.
      Structural basis for expansion of the genetic alphabet with an artificial nucleobase pair.
      ).
      Of the amino acid substitutions in ZP Klentaq, M444V was considered to be the best candidate for providing access to increased relative domain motion as compared with the WT enzyme. Residue 444 resides within the hydrophobic core of the palm domain, which serves as the command center for the enzyme. Both the fingers and thumb domains are connected to the palm domain. In addition, the essential catalytic residues Asp-610 and Asp-785, which coordinate Mg2+ and the incoming dNTP in the active site of the enzyme, reside in the palm domain (Fig. 5F). Substitution of a Val for Met at this position creates open space within the core that could potentially translate into increased motion of the fingers and thumb domains.

      Augmenting crystallography with molecular dynamics simulations of natural and evolved DNA polymerases

      Molecular dynamics (MD) simulations are a well-validated method of studying protein dynamics (
      • Dror R.O.
      • Dirks R.M.
      • Grossman J.P.
      • Xu H.
      • Shaw D.E.
      Biomolecular simulation: a computational microscope for molecular biology.
      ) and have yielded important information about conformational changes undergone by DNA polymerases (
      • Walker A.R.
      • Cisneros G.A.
      Computational simulations of DNA polymerases: detailed insights on structure/function/mechanism from native proteins to cancer variants.
      ,
      • Meli M.
      • Sustarsic M.
      • Craggs T.D.
      • Kapanidis A.N.
      • Colombo G.
      DNA polymerase conformational dynamics and the role of fidelity-conferring residues: insights from computational simulations.
      ). Our recent work also demonstrates that MD simulations can be used to understand how substitutions far from the active site might alter substrate selectivity (
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ). For example, microsecond trajectories have shown how the dynamic motions of ZP Klentaq differ from those of WT Klentaq in the binary complex containing template-primer DNA duplexes (Fig. 6A). They can also highlight correlated motions in networks of residues that are critical to altered domain motions in the variant DNA polymerase (Fig. 6B) (
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ), which permit the engineered enzyme to bind WC and Z:P-containing template-primer duplexes in an equivalent fashion, thereby increasing the efficiency of UBP incorporation. This seems to be a consequence of replacing Met-444 and Asp-551 at the base of the thumb domain by valine and glutamate, respectively, which allows the fingers, palm, and thumb domains in ZP Klentaq to move into position about the AEGIS template-primer duplex. Thus, replacing Met-444 by valine in the hydrophobic core changes the populated interactions with adjacent residues (Phe-564, Met-779, and Leu-780) in the hydrophobic core (Fig. 6C) with the consequence that the G helix becomes more flexible in the variant compared with the WT polymerase (
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ).
      Figure thumbnail gr6
      Figure 6A, two complementary cartoon representations of motions in the binary complexes of WT Klentaq (top) and ZP Klentaq variant (bottom). Left, the major principal component (PC1) (
      • Skjaerven L.
      • Martinez A.
      • Reuter N.
      Principal component and normal mode analysis of proteins; a quantitative comparison using the GroEL subunit.
      ) at early (red), intermediate (white), and late (blue) stages of the sampled MD trajectory, showing that the dynamics of the fingers and palm domains are altered when WC and AEGIS template-primer strands are bound for the two DNA polymerases. Right, domain motions of the polymerase complexed to AEGIS DNA computed using DynDom (
      • Taylor D.
      • Cawley G.
      • Hayward S.
      Quantitative method for the assignment of hinge and shear mechanism in protein domain movements.
      ). For reference, the WT Klentaq/WC DNA binary complex (PDB entry 3SZ2) is shown in gray. Each axis of domain motion is indicated by an arrow, the head of which is colored to represent the moving domain (red, yellow, or purple). The fixed domain (blue) and regions that bend during the motion are shown in green. B, dynamic cross-correlation maps computed with bio3d (
      • Grant B.J.
      • Rodrigues A.P.C.
      • El Sawy K.M.
      • McCammon J.A.
      • Caves L.S.D.
      Bio3d: an R package for the comparative analysis of protein structures.
      ) for the AEGIS DNA-containing binary complex of WT Klentaq (top triangle) and ZP Klentaq (bottom triangle). Correlated (range: 0.25–1) and anti-correlated (range: −0.25 to −1) motions are colored from light to dark blue and pink, respectively. Areas rendered in white correspond to noncorrelated motions (range: −0.25 to 0.25). Secondary structural elements are also included on the map. Colored boxes show interconnected residue networks with altered motions in the two polymerases. C, altered interactions between residue 444 and surrounding side chains in the hydrophobic core observed in the MD trajectories of the WT Klentaq/Watson–Crick DNA binary complex (left), WT Klentaq/AEGIS DNA binary complex (middle left), ZP Klentaq/Watson–Crick DNA binary complex (middle right), and ZP Klentaq/AEGIS DNA binary complex (right). The percentages of occurrence of specific interactions in the MD trajectory are also shown. Based on of Ref.
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ).
      As well as modeling how amino acid substitutions might impact long time-scale motions, such as domain reorientations, MD simulations can illuminate specific molecular changes that have the greatest impact on the altered catalytic biophysical and catalytic properties of the DNA polymerase variant. In the case of ZP Klentaq, the role of each of the substitutions (M444V, P527A, D551E, or E832V) was determined using MD simulations of the four single-point Klentaq variants (
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ). These calculations showed that replacing Met-444 in the hydrophobic core of the palm domain with valine makes a significant contribution to altering dynamic motions in ZP Klentaq. In addition, changing Asp-551 to glutamate alters the interactions of the H and I helices at the base of the thumb domain, which affects G helix flexibility (Fig. 6B) (
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ).
      Care must be taken to perform MD simulations on the correct form of the enzyme. For example, introducing valine in place of Glu-832, which breaks a salt bridge with Arg-596, has little impact on the dynamics of the ZP Klentaq/DNA binary complex. However, it does alter protein/DNA interactions. This substitution may therefore exert its effects in the pre-incorporation (ternary) complex during dNTP binding and incorporation.
      MD simulations have also provided interesting insights into the “closing mechanism” of DNA polymerase I prior to the incorporation of WC nucleobases (
      • Miller 3rd, B.R.
      • Beese L.S.
      • Parish C.A.
      • Wu E.Y.
      The closing mechanism of DNA polymerase I at atomic resolution.
      ) and the temperature-dependent activity of a Klentaq variant in which Ile-707, located 20 Å away from the active site, is replaced by leucine (
      • Modeste E.
      • Mawby L.
      • Miller III, B.
      • Wu E.
      • Parish C.A.
      A molecular dynamics investigation of the thermostability of cold-sensitive I707L Klentaq1 DNA polymerase and its wild-type counterpart.
      ). This I707L Klentaq variant exhibits low activity at 37 °C but efficiently replicates DNA at 68 °C, permitting improved PCR amplification of difficult sequences. MD simulations show that the I707L Klentaq variant is almost immobile at 37 °C but exhibits increased flexibility at 68 °C. In contrast, the WT enzyme exhibits less pronounced dynamic changes in simulations performed at 37 and 68 °C. Thus, it is likely that the reduced activity of the I707L Klentaq variant is associated with the enzyme remaining in its closed conformation at lower temperatures (i.e. dNTP binding is blocked) and that the pre-incorporation complex cannot form. As observed for ZP Klentaq (
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ), the molecular origins of this behavior could be traced to changes in the hydrophobic core; replacing isoleucine by leucine results in an alternate conformation of an adjacent phenylalanine (Phe-749), thereby repositioning the O and O1 helices. As a consequence, the active site becomes filled with nucleobases in the template overhang, thereby blocking entry of the incoming dNTP.

      Outlook

      Realizing the full potential of UBPs in the creation of new research, diagnostic, and therapeutic tools will ultimately depend on our ability to identify DNA polymerase variants that can faithfully and efficiently replicate those UBPs. Many of these applications will necessarily require enzymes that are active at the high temperatures used in PCR, limiting the pool of candidate DNA polymerases primarily to family A and family B members. For other applications, such as using dNaM-dTPT3 (Fig. 1) in semi-synthetic organisms that introduce noncanonical amino acids into specific proteins or the isothermal amplification of AEGIS UBPs (
      • Yang Z.
      • Kim H.-J.
      • Le J.T.
      • McLendon C.
      • Bradley K.M.
      • Kim M.-S.
      • Hutter D.
      • Hoshika S.
      • Yaren O.
      • Benner S.A.
      Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.
      ), DNA polymerases that function at 37 °C, such as those in E. coli, will be adequate as starting points for structure-based engineering or library construction and selection.
      To date, progress in engineering DNA polymerases has been encouraging (
      • Nikoomanzar A.
      • Chim N.
      • Yik E.J.
      • Chaput J.C.
      Engineering polymerases for applications in synthetic biology.
      ). In our work aimed at improving the ability of these enzyme to replicate UBPs (
      • Laos R.
      • Shaw R.
      • Leal N.A.
      • Gaucher E.
      • Benner S.
      Directed evolution of polymerases to accept nucleotides with non-standard hydrogen-bond patterns.
      ,
      • Singh I.
      • Laos R.
      • Hoshika S.
      • Benner S.A.
      • Georgiadis M.M.
      Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide.
      ,
      • Ouaray Z.
      • Singh I.
      • Georgiadis M.M.
      • Richards N.G.J.
      Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
      ), ZP Klentaq takes advantage of increased domain flexibility to improve the replication efficiency of Z:P pairs, even though both WT Klentaq and ZP Klentaq grip the template-primer duplex in a similar manner prior to and following incorporation of the AEGIS UBP.
      Combining X-ray crystallography with MD simulations yields two important conclusions. First, not all substituted residues impact the properties of the evolved enzyme equally. Second, substitution of a key residue, such as Met-444, can result in increased relative domain motion, even in an enzyme that exhibits a very large conformational change during catalysis. It is this altered motion that allows ZP Klentaq to incorporate UBPs more readily than the WT enzyme.
      CSR, CST, and related strategies for the directed evolution of DNA polymerases, which typically produce variants containing several amino acid substitutions, might therefore be improved by targeting fewer residues outside the active site for variation. Coupling structure determinations, MD simulations, and enzymatic characterization will provide guiding principles for CSR library design and facilitate rational approaches to optimize family A DNA polymerases to replicate multiple UBPs. Such work can be guided by understanding amino acid substitutions already identified in Klentaq variants (Table 1); for example, Asn-583, Ile-614, and Met-747 are substituted in three of eight variant enzymes, whereas Glu-520, Glu-602, Ala-609, Glu-615, and Glu-742 are substituted in two of eight variants. None of these residues are involved in direct hydrogen-bonding interactions with the substrate template-primer or the dNTP in the active site (Fig. 7). The past evolutionary history of DNA polymerases may also prove useful for selecting specific sites for variation (
      • Laos R.
      • Thomson J.M.
      • Benner S.A.
      DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides.
      ,
      • Chen F.
      • Gaucher E.A.
      • Leal N.A.
      • Hutter D.
      • Havemann S.A.
      • Govindarajan S.
      • Ortlund E.A.
      • Benner S.A.
      Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection.
      ).
      Figure thumbnail gr7
      Figure 7A cartoon rendering showing two views of the ternary complex of Klentaq (fingers, blue; palm, green; thumb, yellow; exonuclease domain, red) with amino acid substitutions common to evolved Klentaq variants shown in van der Waals sphere renderings (carbon, orange; oxygen, red; nitrogen, blue; sulfur, yellow). None of the side chains of these residues are involved in direct hydrogen-bonding interactions with the template-primer or dNTP in the active site. Most of these residues are distant from substrate template-primer and dNTP, although the main-chain NH of Glu-615 hydrogen-bonds to the 3′-OH of the dNTP deoxyribose ring, and Asn-583 makes a water-mediated hydrogen bond to the backbone of the template strand. In addition, Ile-614 and Glu-615 are within van der Waals contact distance of the dNTP bound to the active site, and Met-747 is within contact distance of the template strand.
      We also note that whereas most engineering efforts to date have focused exclusively on improving the ability of DNA polymerases to incorporate UBPs with little consideration for exonuclease activity, a Thermococcus gorgonarius DNA polymerase variant selected to replicate xeno-nucleic acids contains two amino acid substitutions that inactivate the 3′–5′ exonuclease site (
      • Houlihan G.
      • Arangundy-Franklin S.
      • Porebski B.T.
      • Subramanian N.
      • Taylor A.I.
      • Holliger P.
      Discovery and evolution of RNA and Xna reverse transcriptase function and fidelity.
      ). In theory, incorporation of UBPs that slow down polymerization would allow the substrate to be positioned within the exonuclease active site for proofreading. Whether the exonuclease would efficiently process the newly incorporated unnatural nucleobase or not has yet to be fully investigated. It is possible that engineered DNA polymerases may have to possess optimized exonuclease function as well as improved efficiencies for UBP incorporation to maintain artificial genomes.
      In summary, the next generation of useful DNA polymerases must (i) replicate more than one UBP with rates and fidelities similar to those observed for WT DNA polymerases when replicating WC dNTPs and (ii) exhibit no significant pausing following UBP incorporation to avoid invoking proofreading mechanisms. These properties will permit the fidelity of these engineered enzymes to approach 99.99%, allowing them to generate full-length replication products in applications involving multiple rounds of PCR. We suggest a strategy for obtaining these DNA polymerase variants that builds on studies of ZP Klentaq, in which a combination of X-ray crystallography and computer simulations showed the importance of a few key residues for conferring increased dynamic motion in the thumb and fingers domains, thereby improving UBP incorporation efficiency. In this approach, altering the properties of an evolved DNA polymerase variant by introducing additional amino acid substitutions will take advantage of a fundamental, structure-based understanding of how dynamic changes in residue “networks” impact kinetic properties (
      • Hu S.S.
      • Offenbacher A.R.
      • Lu E.D.
      • Klinman J.P.
      Comparative kinetic isotope effects on first- and second-order rate constants of soybean lipoxygenase variants uncover a substrate-binding network.
      ,
      • Hu S.S.
      • Offenbacher A.R.
      • Thompson E.M.
      • Gee C.L.
      • Wilcoxen J.
      • Carr C.A.M.
      • Prigozhin D.M.
      • Yang V.
      • Alber T.
      • Britt R.D.
      • Fraser J.S.
      • Klinman J.P.
      Biophysical characterization of a disabled double mutant of soybean lipoxygenase: the “undoing” of precise substrate positioning relative to metal cofactor and an identified dynamical network.
      ).

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