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Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA

2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês

10.1074/jbc.m005757200

1083-351X

Premal H. Patel, Lawrence A. Loeb,

CRISPR and Genetic Engineering

DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605–617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 103-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis. DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605–617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 103-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis. DNA polymerase I T. aquaticus wild type polyacrylamide gel electrophoresis Molony murine leukemia virus reverse transcriptase Both DNA and RNA polymerase can catalyze chain elongation reaction guided by single-stranded DNA templates to generate polynucleotide products (1Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman and Co., New York1992Google Scholar). The order of nucleotide addition proceeds in a 5′ → 3′ direction via metal-mediated phosphoryl transfer reaction resulting in the formation of phosphodiester bond and release of pyrophosphate (2Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (760) Google Scholar). In addition, both DNA and RNA polymerases resemble in morphology a cupped human right hand and bind DNA template and the incoming nucleotide within the active site cleft (3Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (451) Google Scholar, 4Sousa R. Chung Y.J. Rose J.P. Wang B.C. Nature. 1993; 364: 593-599Crossref PubMed Scopus (341) Google Scholar). DNA polymerases differ from RNA polymerases in utilizing 2′-deoxynucleotides (dTTP, dCTP, dGTP, and dATP) rather than ribonucleotides (rUTP, rCTP, rGTP, and rATP). A detailed analysis of the polymerase active site is crucial to understanding how these polymerases distinguish between dNTPs and rNTPs, as well as to provide insights on how the two types of polymerases co-evolved to adopt similar mechanisms. Despite the similarity in protein structure and function, there is almost no sequence identity between DNA and RNA polymerases. For example, nearly all of the over 40 prokaryotic and eubacteria DNA pol Is1 sequenced (includingThermus aquaticus pol I, Chlamydia trachomatispol I, and Escherichia coli pol I) contain the DYSQIELR sequence within the dNTP binding site (motif A; Ref. 5Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (86) Google Scholar), yet RNA pols only have in common the catalytically essential aspartic acid residue (6Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (580) Google Scholar). If evolution proceeded from an "RNA world" containing RNA synthesizing enzymes to a "DNA world" with genomes replicated by DNA synthesizing enzymes (7Joyce G.F. Nature. 1989; 338: 217-224Crossref PubMed Scopus (497) Google Scholar, 8Joyce G.F. Curr. Biol. 1996; 8: 965-967Abstract Full Text Full Text PDF Scopus (33) Google Scholar), it might be possible to gain insights into this process by substituting random sequences within the active site of a polymerase. Analyses of high resolution x-ray crystal structures suggest that a single side chain prevents diverse polymerases from incorporating ribonucleotides (9Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (662) Google Scholar, 10Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1107) Google Scholar, 11Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar). Rational approaches involving site-directed mutagenesis of this residue result in polymerases that facilitate ribonucleotide incorporation, but substitutions at this residue could not produce polymerases that efficiently incorporate successive ribonucleotides (12Astatke M. Ng K. Grindle N.D.F. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (182) Google Scholar, 13Gao G. Orlova M. Georgiadis M.M. Hendrickson W.A. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 407-411Crossref PubMed Scopus (165) Google Scholar). A disadvantage of structure-guided site-directed studies is that, by substituting a catalytically important residue to a neutral amino acid, the overall enzyme activity can be impaired. In this report, we offer an alternative to structure based site-directed mutagenesis approach for creating enzymes with unique properties. Assuming that current RNA and DNA polymerases evolved from a common ancestor and share a basic mechanism, it should be possible to evolve one of these enzymes into the other, following extensive rounds of mutagenesis and stringent selection protocols. To explore this concept, we randomly mutated a portion of the active site of a eubacteria DNA polymerase, Taq pol I, selected for functioning mutants, and tested 291 mutant enzymes for the ability to synthesize RNA. Twenty-three different mutant polymerases containing substitutions in predominantly one of two amino acids were identified that incorporated ribonucleotides at a rate approaching 103-fold greater than that of WT Taq DNA polymerase. We show that the active site within several families of WT DNA polymerases is especially evolved to exclude rUTP. The construction of the random Taq pol I library (containing 200,000 individual clones) and genetic selection protocol that yields functional mutants in vivo has been described in detail in a previous publication (5Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (86) Google Scholar). The 350 colonies from the 8000 that complemented pol I temperature sensitive phenotype at 37 °C were isolated and grown in nutrient broth individually overnight at 30 °C. Each culture was grown to A595of 0.3 at 30 °C in 10 ml, and Taq pol I expression was induced with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside and incubations continued for 4 h. Taq pols were partially purified (50 μl total volume) using a modified protocol from Refs. 14Grimm E. Arbuthnot P. Nucleic Acids Res. 1995; 23: 4518-4519Crossref PubMed Scopus (25) Google Scholar and 15Desai U.J. Pfaffle P.K. BioTechniques. 1995; 19 (and 784): 780-782PubMed Google Scholar that allows efficient (>50%) purification of Taq pol I while removing endogenous polymerase and nuclease activities. Biochemical analysis of DNA-dependent DNA polymerase activity of these 350 separate pols showed that 291 pols are active at elevated temperatures (>10% activity relative to WT enzyme). Each of the selected Taq pols that retain at least 10% activity relative to WT enzyme at 72 °C (291 total) were tested for the ability to incorporate ribonucleotides. Primer-template constructs were prepared by hybridizing 5′-32P end-labeled 23-mer primer (5′-cgc gcc gaa ttc ccg cta gca at) with 46-mer template (5′-gcg cgg aag ctt ggc tgc aga ata ttg cta gcg gga att cgg cgc g) using a 1:2 primer:template ratio (16Patel P.H. Preston B.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 549-553Crossref PubMed Scopus (143) Google Scholar). The primer/template (5 nm) was incubated in a reaction mixture containing 50 mm KCl, 10 mm Tris-HCl (pH 8), 0.1% Triton X-100, 2.5 mmMgCl2, and 1 μl of partially purified Taq pols (0.1 to 0.01 units) in 10-μl volumes in the presence of 0–250 μm each rNTP. Reactions were terminated after 30 min of incubation at 55 °C with the addition of 2 μl of formamide containing stop solution (Amersham Pharmacia Biotech). Products were analyzed by 14% denaturing PAGE (16Patel P.H. Preston B.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 549-553Crossref PubMed Scopus (143) Google Scholar). Incorporation of ribonucleotides relative to deoxyribonucleotides onto nascent primers results in a slower migration on PAGE gels; only mutants that produced products with such migration patterns were considered rNTP incorporating polymerases. The RNA polymerase activity for several of these enzymes was confirmed by assaying for activity in a 20-μl reaction mixture containing 50 mm KCl, 10 mm Tris-HCl (pH 8), 0.1% Triton-X, 2.5 mm MgCl2, 0.4 mg of activated calf thymus DNA, 10 μm each dCTP, dATP, and dTTP, 500 μm rGTP, 0.25 mCi of [α-32P]dTTP, and 1 μl of WT or mutant Taq pols (0.1 units). Incubations were at 72 °C for 5 min, and reactions were stopped with the addition of 100 μl 0.1 m sodium pyrophosphate, followed by 0.5 ml of 10% trichloroacetic acid. Polymerase activity was quantified by collecting precipitated radioactive DNA onto glass filter papers, and the radioactive counts were measured by scintillation. Wild type and mutant (mutants 53, 65, 75, 94, 230, 265, 300, and 346) Taq pols were purified to homogeneity (>90% purity) using a modified procedure (17Engelke D.R. Krikos A. Bruck M.E. Ginsburg D. Anal. Biochem. 1990; 191: 396-400Crossref PubMed Scopus (136) Google Scholar). For step 1, bacteria cultures (DH5α cells; 2 liters) harboring pTaq or selected mutant pTaqLIB plasmid were harvested and lysed in the presence of buffer A (30 mm Tris-HCl, pH 7.9, 50 mm glucose, 1 mm EDTA, 0.5 mmphenylmethylsulfonyl fluoride, 0.5% Tween 20, 0.5% Nonidet P-40) with lysozyme (4 mg/ml) by freezing and thawing at −70 °C and 70 °C. For step 2, Taq pol was precipitated by the addition of polyethyeneimine at a final concentration of 0.1%, recovered by centrifugation, washed with buffer containing low salt (0.025m KCl) buffer C (20 mm HEPES, pH 7.9, 1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.5% Tween 20, 0.5% Nonidet P-40), and then solubilized in 0.15m KCl buffer C. For step 3, the enzyme was diluted to 50 mm KCl and loaded onto a pre-equilibrated HiTrap Heparin 5-ml column at 1 ml/min flow rate. The column was washed with 10 volumes of Buffer C (50 mm KCl), and the protein was eluted using a linear gradient from 50 to 750 mm KCl (60 ml). Fractions (1 ml) were assayed for polymerase activity by measuring incorporation of [α32P]dGTP at 70 °C using activated calf thymus DNA as a template with Mg2+ and all four dNTPs including [α32P]dGTP. Peak fractions with WT and mutant enzymes consistently eluted at approximately 300 mm KCl, were >90% pure upon analysis by SDS-PAGE, and were stored in 20% glycerol at −70 °C. Klenow DNA polymerase I (3′-5′ exo− or 3′-5′ exo+) and Vent polymerase (3′-5′ exo−) were obtained from New England Biolabs. MMLV RT (RNaseH−; SuperScript II) was obtained from Life Technologies, Inc. A 47-mer template (3′-gcg cgg ctt aag ggc gat cgt tat agc tta agg cct tta aag ggc cc-5′) was hybridized with one of four primers: 23-mer (5′-cgc gcc gaa ttc ccg cta gca at), 24-mer (5′-cgc gcc gaa ttc ccg cta gca ata), 25-mer (5′-cgc gcc gaa ttc ccg cta gca ata t), or 26-mer (5′-cgc gcc gaa ttc ccg cta gca ata tc). All single nucleotide incorporation with dATP or rATP required 23-mer/47-mer primer/template; reactions with dTTP, dUTP, or rUTP required 24-mer/47-mer; reactions with dCTP or rCTP required 25-mer/47-mer; those with dGTP or rGTP required 26-mer/47-mer. The steady state Michaelis-Menten parameters Vmaxand Km were determined following incubations with limiting amounts of Taq pol in the presence of 5 nm primer/template and varying concentration of each dNTP or rNTP for 10 min at 55 °C for reactions containing Taqpol I or at 37 °C for reactions containing other polymerases as described in Ref. 18Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14699Abstract Full Text PDF PubMed Google Scholar. The kinetic rate parameterkcat was calculated by dividingVmax by the enzyme concentration. All products were analyzed by 14% PAGE and quantified by phosphorimager analysis. Polymerization reactions in the presence of all four rNTPs were conducted with three template sequences: 47-mer 5′-ccc ggg aaa ttt ccg gaa ttc gat att gct agc ggg aat tcg gcg cg-3′; 46-mer 5′-gcg cgg tcg ctt ggc tgc cgt cta ttg cta gcg gga att cgg cgc g; and a template corresponding to the 3′ terminal 125 nucleotides encoding Taq polymerase. Templates were hybridized with 5′-32P-labeled 23-mer primer (5′-cgc gcc gaa ttc ccg cta gca at) to form either 23-mer/47-mer double-stranded DNA (primer-template), 23-mer/46-mer double-stranded DNA, or DNA corresponding to the 3′ terminus of T. aquaticus polA gene as described (16Patel P.H. Preston B.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 549-553Crossref PubMed Scopus (143) Google Scholar). Polymerizations were conducted in vitroat 55 °C for 5–30 min in 10–40-μl reaction mixtures containing 5 nm primer-template, 50 mm KCl, 10 mm Tris-HCl (pH 8), 0.1% Triton-X, 2.5 mmMgCl2, all four rNTPs (50–500 μm each), and either WT or mutant Taq pol I (2–20 nm). Reactions were initiated with the addition of enzyme and stopped with the addition of sequencing loading buffer (Amersham Pharmacia Biotech). We measured the capacity of several subclasses of DNA polymerases to incorporate ribonucleotide triphosphates relative to dNTPs. DNA polymerases from the thermophile eubacterium T. aquaticus, from hyperthermophile archaea Thermococcus litoralis, from prokaryote E. coli, and from retrovirus MMLV were analyzed. These DNA polymerases represent three major subclasses (6Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (580) Google Scholar): prokaryotic pol I family (Taq pol I and E. coli pol I), mammalian pol α family (Vent pol), and RT (MMLV RT). Incorporation of individual ribonucleotides by WT Taq pol I requires greater amounts of polymerase and at least 1000-fold higher concentrations of each rNTP relative to the corresponding dNTP (Fig. 1). It is important to note that polynucleotide products containing a 3′ ribonucleotide migrate slower within polyacrylamide gels relative to 3′ deoxynucleotide products. This property is useful to monitor the nature of products, as well as to ensure the purity of dNTP and rNTP substrates. Hyperbolic curve fit of steady state rates plotted as a function of nucleotide concentration yields Michaelis-Menten parameters Vmax andKm, and the initial slope of this plot reflects the catalytic efficiency of each enzyme. WT Taq pol I incorporates dGTP, dATP, and dCTP up to 30,000 times more efficiently (kcat/Km) than the respective ribonucleotides (Tables I and III). The low efficiency of rNTP incorporation is largely due to a 1000-fold greater Km for rG, rC, and rA relative to the respective deoxynucleotides. Other DNA polymerases from pol I, pol α, and RT families also incorporate ribonucleotides inefficiently (also largely because of high Km for rNTPs relative to dNTPs; Table I).Table IEfficiency of dNTP and rNTP incorporation opposite complementary bases by diverse polymerasesProteinNucleotideVmax(rel)KmVmax/KmDiscrimination 1-aDiscrimination represents the efficiency (Vmax/Km) of dNTP incorporation divided by efficiency of the corresponding rNTP. For dUTP, discrimination represents efficiency of dTTP incorporation divided by that of dUTP. Vmax (rel) represents steady state rates relative to that of dTTP or dCTP incorporation.μmTaqpoldTTP 1-bPrimer/template sequence used with each nucleotide is defined under "Experimental Procedures."1.00.005200dUTP1.10.0071601.2rUTP0.032400.000131,500,000dCTP1.00.01224rCTP0.06590.00124,000Klenow (3′-5′ exo−)dTTP1.00.01663dUTP1.10.028391.6rUTP0.030600.00050130,000dCTP1.00.04025rCTP0.20250.0083,100VentdTTP1.00.03926dUTP1.040.055191.4rUTP0.079310.002510,000dCTP1.01.10.90rCTP0.0791800.000442,000MMLV RTdTTP1.00.05419dUTP0.620.213.06.3rUTP0.035390.0009021,000dCTP1.00.215.0rCTP0.054130.00421,100All values have ±<10% error.1-a Discrimination represents the efficiency (Vmax/Km) of dNTP incorporation divided by efficiency of the corresponding rNTP. For dUTP, discrimination represents efficiency of dTTP incorporation divided by that of dUTP. Vmax (rel) represents steady state rates relative to that of dTTP or dCTP incorporation.1-b Primer/template sequence used with each nucleotide is defined under "Experimental Procedures." Open table in a new tab Table IIIEfficiency of dNTP and rNTP incorporation by WT and several mutant Taq pol IProteinNucleotidedNTPrNTPdNTP/rNTP discrimination 3-adNTP/rNTP discrimination equals efficiency of dNTP incorporation (kcat/Kn) relative to rNTP incorporation.kcatKnkcat/KmkcatKnkcat/Kns−1μms−1μmWild typeG 3-bPrimer/template sequence used with each nucleotide is defined in the "Methods" section.0.0200.0211.00.0026763.5 × 10−529,000A0.0120.0700.170.00162307.0 × 10−624,000C0.0130.0420.310.00083591.4 × 10−522,000T/U0.0130.00502.60.000432401.8 × 10−61,400,000Mutant 53 (I614K)G0.0790.370.210.0969.00.01119A0.0350.110.320.035120.0029110C0.0790.0830.950.067100.0067140T/U0.0460.0311.50.0962700.000364,200Mutant 94 (A608S, I614N)G0.00580.0860.0670.00650.940.007010A0.0120.150.0800.00656.70.0009783C0.00580.0890.0650.0075140.00054120T/U0.00710.0220.320.0067310.000221,500Mutant 265 (I614N, L616I)G0.0150.00712.10.0120.770.016130A0.0140.0480.290.00804.40.0018160C0.0150.0340.440.00736.20.0012370T/U0.0160.0161.00.017350.000492,100Mutant 346 (A608D, E615D)G0.00200.120.0170.00561.70.00325.3A0.00400.200.0200.0040260.00015130C0.00200.290.00690.00365.40.0006710T/U0.00870.0180.48All values have ±<10% error.3-a dNTP/rNTP discrimination equals efficiency of dNTP incorporation (kcat/Kn) relative to rNTP incorporation.3-b Primer/template sequence used with each nucleotide is defined in the "Methods" section. Open table in a new tab All values have ±<10% error. All values have ± 1000-fold higher Km for these rNTPs relative to the respective dNTPs, and the WT enzyme exhibits ≈50,000-fold higherKm for rUTP relative to dTTP or dUTP. Overall, the mutants incorporate each ribonucleotide up to 3 orders of magnitude more efficiently than the WT polymerase (Table III; dNTP/rNTP Discrimination). Mechanistically, the ability of the mutant polymerases to efficiently incorporate ribonucleotide results from a >10-fold faster incorporation rate relative to WT enzymes and the ability to incorporate rNTPs at concentrations that are significantly below physiologic levels. The relatively fast catalytic rate of rNTP incorporation, coupled with the Km for rNTP significantly lower than the in vivo ribonucleotide concentration (approximately 1000 μm each), suggest these enzymes should be able to incorporate ribonucleotides at physiologic conditions. To determine whether these polymerases can function as RNA polymerases by incorporating multiple ribonucleotides sequentially, we incubated purified WT Taq pol I, a mutant containing a substitution at Ile-614, and a mutant containing a substitution at Glu-615 in the presence of increasing amounts of all four rNTPs (Fig. 2). Although the WT enzyme inefficiently incorporates and extends ribonucleotides, both rNTP utilizing mutant enzymes polymerize multiple ribonucleotides, even at rNTP concentrations below that found in cells. Interestingly, the strong pause sites produced at runs of template dAs is exactly what one would predict from the kinetic data (Tables I and III), demonstrating a specific decrease in incorporation (and perhaps a decreased extension of 3′-rU sequences). Extension past these runs is facilitated by either increasing incubation time or increasing ribonucleotide concentration. In the presence of a trace amount of a metal cofactor Mn+2(0.5 mm Mn+2 and 2.5 mmMg+2), this pause site is markedly reduced, and elongation proceeds up to the 5′ end of the template even in presence of low rNTP concentrations. Elongated RNA products are also efficiently generated by mutant polymerases containing substitutions at either Ile-614 or Glu615 using templates containing minimal template dA residues. Using such a template, RNA polymerization to the 5′ template terminus by specific mutant polymerases occurs in a time-dependent manner and within minutes of incubation (Fig. 3). RNA synthesis is inefficient with the WT Taq pol I even after prolonged incubation times, and the limited products formed by WT enzyme are resistant to alkali degradation. In contrast, addition of alkali degraded elongated products synthesized by mutant enzymes to regenerate the initial substrate, illustrating the products are RNA (Fig. 2, lane 32 C, and Fig. 3, lanes OH). From the time-dependent accumulation of products, we estimate that the mutant Taq pols can synthesize extended RNA products (containing >10 nucleotides) at a >1000-fold faster rate relative to WT Taq pol I. The ability to synthesize long RNA products correlates with the DNA polymerase activity of the mutant enzyme. For example mutant 94 (A608S,I614N) with low DNA polymerase activity as measured on activated calf thymus DNA does not efficiently synthesize long stretches of RNA. Mutant Taq pol Is exhibiting high DNA polymerase activity (mutants 75 (I614M), 265 (I614N,L616I), and 346 (A608D,E615D)) can synthesize nucleic acids containing as many as 100 successive ribonucleotides on templates with limited dA residues (data not shown). To our knowledge, this is the first report of a highly active polymerase that can efficiently synthesize long stretches of either DNA or RNA.Figure 3RNA synthesis. A 5′-32P-labeled 23-mer primer (5′-cgc gcc gaa ttc ccg cta gca at) was hybridized with 46-mer (5′-gcg cgg tcg ctt ggc tgc cgt cta ttg cta gcg gga att cgg cgc g) template, and polymerization reactions were conducted at 55 °C in the presence of 500 μm each of the four rNTPs, 2.5 mm Mg Cl2, 0.5 mm MnCl2, and 2 fmol/μl of either WTTaq pol I, mutant 53 (I614K), mutant 75 (I614M), mutant 265 (I614N and L616I), and mutant 346 (A608D and E615D). Incubations (40 μl) were stopped by removing 10 μl of the incubation sample after 5, 15, and 30 min into formamide containing stop solution; the remaining 10 μl was further incubated in the presence of 1.0n NaOH for 10 min at 55 °C (lane sOH) prior to adding stop solution. The migration pattern of product bands by WTTaq pol I is not the same as that with mutant enzyme upon varying exposure intensity, and products formed by WT enzyme are resistant to alkali hydrolysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There are two major conclusions from this study. First, DNA polymerases selectively exclude rUTP. Second, amino acid substitutions at two different positions within the DNA polymerase catalytic site can result in enzymes that incorporate ribonucleotides and synthesize RNA. These findings were not predicted by analysis of high resolution x-ray crystal structures. These observations link DNA and RNA polymerization mechanisms and may provide insights into the evolution of DNA polymerases during the postulated progression form an RNA into DNA world (7Joyce G.F. Nature. 1989; 338: 217-224Crossref PubMed Scopus (497) Google Scholar, 8Joyce G.F. Curr. Biol. 1996; 8: 965-967Abstract Full Text Full Text PDF Scopus (33) Google Scholar). Taq pol I incorporates rUTP 1,000,000-fold less efficiently relative to dTTP or dUTP (Tables I and III). Joyce and co-workers (12Astatke M. Ng K. Grindle N.D.F. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (182) Google Scholar) also showed that WT E. coli pol I discriminates against rUTP relative to dTTP by 106-fold. We extended this finding by comparing dNTP relative to rNTP incorporation by DNA polymerases from different families. Our results indicate that ribouracil triphosphate exclusion is a general property of many DNA polymerases. With WTTaq pol I, the 106-fold difference in nucleotide incorporation efficiency between dTTP or dUTP relative to rUTP incorporation is the highest discrimination factor that we have encountered with natural substrates. Although the concentration of each deoxynucleotide including dTTP is 25–100 μm in organisms, that of each ribonucleotides, including rUTP, is approximately 1000 μm (19Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1328) Google Scholar). We estimate that if WTTaq pol I were to replicate the entire 2 × 106 base pair eubacteria genome (20Moreira L.M. Da Costa M.S. Sa-Correia I. Arch. Microbiol. 1997; 2: 92-102Crossref Scopus (8) Google Scholar), assuming the rUTP concentration is 10-fold greater than that of dTTP, WT Taqpol I should introduce approximately 10 rUMP into genomic DNA per replication cycle, and these substitutions would presumably be corrected by DNA repair. Other polymerases from prokaryotic pol I, eukaryotic pol α, and RT families also discriminate against ribouracil (Table I). This DNA polymerase property of specifically discriminating against ribouracil might have resulted from natural selection. Cells have evolved mechanisms to exclude uracil from DNA. When formed by deamination of cytadines (21Lindahl T. Nyberg B. Biochemistry. 1974; 13: 3405-3410Crossref PubMed Scopus (588) Google Scholar), uracil is a potent promutagen inducing G·C→A·T transition mutations (22Duncan B.K. Weiss B. J. Bacteriol. 1982; 2: 750-755Crossref Google Scholar). Uracil lesions are also formed by the incorporation of dUTP by DNA polymerases because dUTP can readily substitute for dTTP (Table I and Ref. 23Olivera B.M. Manlapaz-Ramos P. Warner H.R. Duncan B.K. J. Mol. Biol. 1979; 128: 265-275Crossref PubMed Scopus (13) Google Scholar). Removal of uracil residues in DNA is accomplished by uracil-DNA glycosylase, and repair synthesis involves the generation of single-stranded breaks, a potential source of mutagenesis (24Bonura T. Schultz R. Friedberg E.C. Biochemistry. 1982; 10: 2548-2556Crossref Scopus (11) Google Scholar, 25Slupphaug G. Mol C.D. Kavli B. Arvai A.S. Krokan H.E. Tainer J.A. Nature. 1996; 384: 87-92Crossref PubMed Scopus (485) Google Scholar). In cells, rUTP is present in millimolar concentrations, well in excess of dTTP and dUTP concentrations (on average, 40 and 0.2 μm, respectively; Refs. 19Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1328) Google Scholar and 23Olivera B.M. Manlapaz-Ramos P. Warner H.R. Duncan B.K. J. Mol. Biol. 1979; 128: 265-275Crossref PubMed Scopus (13) Google Scholar) and thus can be a potent source of damage (e.g. single-stranded breaks) if introduced into DNA in amounts that exceed the capacity of the cell for DNA repair. Analysis of a high resolution crystal structure of Taq pol I bound to DNA and dNTP (9Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (662) Google Scholar) suggested that a single residue can sterically exclude the 2′-OH of an incoming rNTP (Fig. 4). This glutamic acid residue is conserved in over 35 prokaryotic and eubacteria pol I sequences, including that of E. coli pol I (GenBankTM; Ref. 5Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (86) Google Scholar). Polymerases in the pol α and RT families contain a planar ringed amino acid (usually Tyr or Phe) at this locus; within the HIV-1 RT structure bound with DNA and dNTP the homologous Tyr-115 residue is positioned adjacent to the incoming nucleotide and likely also prevents incorporation of ribonucleotides by sterically interfering with 2′-OH group (11Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1360) Google Scholar, 26Bonnin B. Lazaro J.M. Blanco L. Salas M. J. Mol. Biol. 1999; 1: 241-251Crossref Scopus (75) Google Scholar). Modeling the selected substitutions into WTTaq pol I structure, followed by energy minimization suggests there are at least two mechanisms by which the steric interference conferred by Taq pol I Glu-615 with the 2′-OH group of the incoming ribonucleotide can be alleviated. First, modeling studies in Taq pol I suggests alterations that reduce the length of the Glu-615 side chain should permit ribonucleotide binding (Fig. 4 and Table III). Joyce and co-workers (12Astatke M. Ng K. Grindle N.D.F. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (182) Google Scholar) demonstrated that E710A substitution within E. coli pol I (Klenow), at a site identical to Taq pol I Glu-615 residue, permits ribonucleotide incorporation. We find substitutions to aspartic acid for Taq pol I Glu-615 facilitates ribonucleotide incorporation. So far, we have not yet detected a Glu → Ala substitution in the Taq pol I motif A active mutant library, as well as within an extensive motif A mutant library of E. coli pol I, presumably because of reduced catalytic activity of mutants not containing either Asp or Glu at this site (5Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (86) Google Scholar). 2P. H. Patel and L. A. Loeb, J. Biol. Chem., in press.. Second, we find that diverse substitutions at the adjacent residue Ile-614 allow ribonucleotide incorporation and that evolved enzymes containing substitutions at position 614 utilize rNTPs at efficiencies equivalent to enzymes containing E615D substitutions (Fig. 3 and Table III). Analysis of Taq pol I structure model bound with DNA and a rNTP shows the ribose ring of the rNTP packs closely against Ile-614. Residue Ile-614 is located at a junction of a highly conserved β strand and α helix and is highly mutable. Energy minimizations of models of substitutions at this residue, which confers the ability to incorporate rNTPs, suggest that these substitutions cause this junction to be located further from the incoming nucleotide, thus allowing 2′-OH to fit. In summary, we find that motif A within the DNA polymerase active site is highly plastic and can tolerate numerous substitutions while preserving physiologic DNA polymerase activity, and we have used this flexibility in structure to evolve a set of enzymes with altered substrate specificity. Mutant polymerases that can synthesize both DNA and RNA may be useful for biotechnology and allow automated coupled polymerase chain reaction amplification and transcription by cycling nucleotide triphosphates within the reaction mixture. These polymerases could also be used for DNA sequence analysis, following specific incorporation of a ribonucleotide during polymerase chain reaction and subsequent alkali cleavage at the position of ribosubstitution (27Barnes W.M. J. Mol. Biol. 1978; 119: 83-99Crossref PubMed Scopus (28) Google Scholar). In addition, these polymerases allow one to introduce nucleotide analogs containing adducts (e.g. fluorophores) attached to the 2′ ribose ring. The plasticity of the DNA polymerase active site should facilitate evolution within the laboratory of other polymerases for the incorporation of specific nucleotide analogs that are of mechanistic or medical importance. We thank Dr. Ellie Adman for Taqpol I structure illustrations, calculations, and discussions.