Skip to main content
  • Submit
  • About
    • Editorial Board
    • PNAS Staff
    • FAQ
    • Accessibility Statement
    • Rights and Permissions
    • Site Map
  • Contact
  • Journal Club
  • Subscribe
    • Subscription Rates
    • Subscriptions FAQ
    • Open Access
    • Recommend PNAS to Your Librarian
  • Log in
  • My Cart

Main menu

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses
  • Submit
  • About
    • Editorial Board
    • PNAS Staff
    • FAQ
    • Accessibility Statement
    • Rights and Permissions
    • Site Map
  • Contact
  • Journal Club
  • Subscribe
    • Subscription Rates
    • Subscriptions FAQ
    • Open Access
    • Recommend PNAS to Your Librarian

User menu

  • Log in
  • My Cart

Search

  • Advanced search
Home
Home

Advanced Search

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses

New Research In

Physical Sciences

Featured Portals

  • Physics
  • Chemistry
  • Sustainability Science

Articles by Topic

  • Applied Mathematics
  • Applied Physical Sciences
  • Astronomy
  • Computer Sciences
  • Earth, Atmospheric, and Planetary Sciences
  • Engineering
  • Environmental Sciences
  • Mathematics
  • Statistics

Social Sciences

Featured Portals

  • Anthropology
  • Sustainability Science

Articles by Topic

  • Economic Sciences
  • Environmental Sciences
  • Political Sciences
  • Psychological and Cognitive Sciences
  • Social Sciences

Biological Sciences

Featured Portals

  • Sustainability Science

Articles by Topic

  • Agricultural Sciences
  • Anthropology
  • Applied Biological Sciences
  • Biochemistry
  • Biophysics and Computational Biology
  • Cell Biology
  • Developmental Biology
  • Ecology
  • Environmental Sciences
  • Evolution
  • Genetics
  • Immunology and Inflammation
  • Medical Sciences
  • Microbiology
  • Neuroscience
  • Pharmacology
  • Physiology
  • Plant Biology
  • Population Biology
  • Psychological and Cognitive Sciences
  • Sustainability Science
  • Systems Biology
Research Article

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

Samra Obeid, Anna Baccaro, Wolfram Welte, Kay Diederichs, and Andreas Marx
PNAS December 14, 2010 107 (50) 21327-21331; https://doi.org/10.1073/pnas.1013804107
Samra Obeid
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Anna Baccaro
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wolfram Welte
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kay Diederichs
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andreas Marx
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: andreas.marx@uni-konstanz.de
  1. Edited by Jack W. Szostak, Massachusetts General Hospital, Boston, MA, and approved October 29, 2010 (received for review September 14, 2010)

  • Article
  • Figures & SI
  • Info & Metrics
  • PDF
Loading

Abstract

Numerous 2′-deoxynucleoside triphosphates (dNTPs) that are functionalized with spacious modifications such as dyes and affinity tags like biotin are substrates for DNA polymerases. They are widely employed in many cutting-edge technologies like advanced DNA sequencing approaches, microarrays, and single molecule techniques. Modifications attached to the nucleobase are accepted by many DNA polymerases, and thus, dNTPs bearing nucleobase modifications are predominantly employed. When pyrimidines are used the modifications are almost exclusively at the C5 position to avoid disturbing of Watson–Crick base pairing ability. However, the detailed molecular mechanism by which C5 modifications are processed by a DNA polymerase is poorly understood. Here, we present the first crystal structures of a DNA polymerase from Thermus aquaticus processing two C5 modified substrates that are accepted by the enzyme with different efficiencies. The structures were obtained as ternary complex of the enzyme bound to primer/template duplex with the respective modified dNTP in position poised for catalysis leading to incorporation. Thus, the study provides insights into the incorporation mechanism of the modified nucleotides elucidating how bulky modifications are accepted by the enzyme. The structures show a varied degree of perturbation of the enzyme substrate complexes depending on the nature of the modifications suggesting design principles for future developments of modified substrates for DNA polymerases.

  • modified nucleotide
  • nucleobase modification
  • functional DNA
  • X-ray structure
  • PCR

The capability of DNA polymerases to accept modified 2′-deoxynucleoside triphosphates (dNTPs) is exploited in many important biotechnological applications. These include next-generation sequencing approaches (1–3), single molecule sequencing (4), labeling of DNA and PCR amplificates, e.g., for microarray analysis (5–8), DNA conjugation (9), or the in vitro selection of ligands such as aptamers by SELEX (systematic enrichment of ligands by exponential amplification) (10). Furthermore, utilizing the intrinsic properties of DNA in combination with chemically introduced functionalities provides an entry to new classes of nucleic acids-based hybrid materials (9). In most cases the dNTP modifications were introduced to the nucleobase. In cases where pyrimidines are employed the modifications are almost exclusively at the C5 position to avoid disturbing of Watson–Crick base pairing ability (11–20). In addition, the C5 modification is well accommodated into the major groove of DNA without perturbing the DNA structure. Mainly C5 alkyne modified dNTPs are used because they are accessible via metal mediated cross coupling reactions (21–23) in a straightforward manner. While many C5 modified dNTP analogs are already applied, because they are processed by DNA polymerases at least to some extent, it is still unpredictable which modification will be accepted (8, 11–23) because the detailed molecular mechanism by which C5 modifications are accepted by a DNA polymerase is not at all understood. This is largely due to the absence of suitable structural data.

Here, we present unique crystal structures of a DNA polymerase caught while processing two different C5 modified dNTPs, respectively. The large fragment of Thermus aquaticus (Taq) DNA polymerase (in short KlenTaq, N-terminally truncated form of Taq polymerase) was chosen as the target because this enzyme class is heavily employed and well characterized on a functional and structural level (24–31). We compared two dNTP analogs—namely dTspinTP and dTdendTP (Fig. 1A). It was already shown that these nucleotides are able to replace natural dTTP in DNA polymerase-catalyzed reactions (22, 23). In the case of dTspinTP, the nitroxide modification is attached to the C5 position of the nucleobase via a rigid and short acetylene linker that is to ensure direct monitoring of DNA dynamics by EPR (electron paramagnetic resonance) spectroscopy. The dendron-modified substrate dTdendTP is a flexible and sterically demanding modification. The branched architecture that is connected to the nucleobase via a propargylamide linker is considered to explore the size limits of modifications tolerated by DNA polymerases and to introduce multiple functional groups in a single incorporation step. In this study we employed these analogs as models for C5 modified dNTPs because their structure resembles those dNTPs used for other purposes.

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Comparison of dTspinTP and dTdendTP in single incorporation experiments. (A) Chemical structure of dTspinTP and dTdendTP. (B) Single nucleotide incorporation of dTRTP for 5, 10, 30, 60, 120, 180, or 300 sec, respectively, using KlenTaq. The respective dTRTP is indicated. In each case, the first lane shows the labeled primer. (C) Competition experiment: single nucleotide incorporation in presence of dTspinTP and dTdendTP. The ratio of dTspinTP/dTdendTP was varied from 1/1 to 100/1 (1/1, 2/1, 4/1, 10/1, 20/1, 50/1, 100/1). The first lane shows the labeled primer.

Results

Single Nucleotide Incorporation of C5 Modified dNTPs.

To investigate the acceptance of the modified nucleotides by KlenTaq primer extension studies of single nucleotide incorporation opposite a templating dA was conducted with dTTP and the two modified thymidine analogs in a time-dependent manner. The reaction products of the modified dTRTP migrate slower than the elongated primer in the presence of dTTP in denaturing polyacrylamide gel electrophoresis (PAGE) due to the additional bulk of the modification (Fig. 1B). Similar effects have been reported before (17, 22, 23). Interestingly, these experiments (Fig. 1B) show the preferential incorporation of the sterically more demanding dTdendTP compared to dTspinTP. The competition experiment, which is performed in present of the two modified triphosphates with an increasing ratio of dTspinTP, shows more obviously that KlenTaq favors dTdendTP over dTspinTP (Fig. 1C).

Quantification of these findings by pre-steady-state kinetics confirmed that indeed dTdendTP is about 20 times more efficiently (kpol/KD) incorporated compared to dTspinTP (Table 1 and Figs. S1 and S2). Interestingly, the smaller dTspinTP shows higher binding affinity (apparent KD) to the enzyme as compared to dTdendTP, whereas its incorporation rate (kpol) is significantly more impaired.

View this table:
  • View inline
  • View popup
Table 1.

Transient kinetic parameters for nucleotide incorporation by KlenTaq wild type

Structure of KlenTaq in Complex with DNA and C5 Modified dNTP.

To reveal the structural basis for the ability of DNA polymerases to process an artificial substrate we crystallized KlenTaq as ternary complex bound to a primer/template duplex and a respective C5 modified triphosphate in the incoming position. The crystals were obtained by a strategy previously used for KlenTaq bound to unmodified substrates (24–26, 28). By incorporation of ddCMP at the primer terminus a primer/template duplex lacking a 3′-terminal hydroxyl group is formed to terminate the primer extension reaction. The structures were solved by difference Fourier techniques and provide snapshots of incorporation of C5 alkyne modified triphosphates at resolutions of 1.9–2.0 Å (Table S1).

Structure of KlenTaq in Complex with DNA and dTspinTP.

KlenTaq in complex with spin-labeled triphosphate dTspinTP (henceforth termed KlenTaqspin) adopted an overall conformation very similar to the one in a ternary complex of the enzyme bound to DNA and ddTTP (PDB ID: 1QTM, henceforth termed KlenTaq1QTM) described earlier (rmsd for Cα 0.392 Å, Fig. S3A and Fig. S4). In KlenTaq1QTM ddTTP was bound to the active site due to the applied crystallization strategy (24–26, 28). A zoom into the active site shows that the dTspinTP is located opposite to adenine of the template strand, forming Watson–Crick base pairing interactions to the templating base (Fig. 2A) and undergoes π stacking interactions to the 3′-terminus of the primer strand (Fig. S5). The distance from the primer 3′-terminus to the α-phosphate of dTspinTP is slightly larger (0.5 Å) than the one in KlenTaq1QTM (Fig. 2C). Furthermore, the O helix of the finger domain packs against the nascent base pair forming an active and closed complex similar as with ddTTP. However, Arg660, located at the N terminus of the O helix, shows a different orientation than in KlenTaq1QTM and is positioned in a way to make room for the nitroxide modification (Fig. 2). Arg660 has been suggested to stabilize the closed and active conformation via hydrogen-bonding interaction to the phosphate backbone of the primer 3′-terminus (26, 28). This interaction is exclusively observed in the closed conformation and may act as a clamp between the finger domain and the bound DNA duplex (26, 28). The interaction of Arg660 with the primer 3′-terminus is hindered by the rigid nitroxide modification connected to the nucleobase and thereby prevents the stabilization of the closed conformation. As a result of the loss of the hydrogen-bonding interaction, Arg660 shows a high flexibility (B-factor of the amino acide side chain is 81 Å2), and the O helix is slightly shifted (Fig. S6). The missing clamp, between the finger domain and the primer/template duplex, may account for the observed 2,500-fold decrease in incorporation efficiency of dTspinTP compared with the natural dTTP.

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Structure of KlenTaq in complex with dTspinTP (KlenTaqspin). (A) The close-up view of KlenTaqspin (blue) shows the incoming dTspinTP (magenta), the O helix with amino acid side chain R660, the primer and template strand. The dashed line highlights the Watson–Crick base pairing interactions. (B) Superimposition of KlenTaqspin and KlenTaq (green, PDB: 1QTM) highlights the different orientation of the amino acid side chain R660. (C) The distance (shown in dashed lines) of the α-phosphate to the primer 3′-terminus is indicated for KlenTaqspin. (D) The distance of the α-phosphate to the primer 3′-terminus (dashed line; 3.5 Å) and the interaction of R660 with the phosphate-backbone of the primer 3′-terminus is indicated in dashed line (3.4 Å) for KlenTaq. No such interaction is found in KlenTaqspin (see C) due to displacement of R660 by the modification.

Structure of KlenTaq in Complex with DNA and dTdendTP.

The structure of KlenTaq in complex with primer/template duplex and dendron-modified triphosphate dTdendTP (henceforth termed KlenTaqdend) was determined with a resolution of 1.9 Å. In comparison with KlenTaq1QTM the overall conformation of KlenTaqdend is very similar, resulting in a low rmsd for Cα 0.250 Å (Fig. 4 and Fig. S3B). The dTdendTP is captured in the active site waiting for insertion (Fig. 3A). We observe a closed and active complex very similar to KlenTaq1QTM and KlenTaqspin (Fig. 3B). The same stabilizing factors that are described above such as Watson–Crick base pairing and π stacking interaction are observed (Fig. S5). Even the distance from the primer 3′-terminus to the α-phosphate of the dTdendTP is similar to the observed distance in KlenTaq1QTM (Fig. 3C). Only partial electron density could be observed for the dendron structure due to the high flexibility of the entity (Fig. 3D and Fig. S4). The rigid propargylamide linkage is well defined, whereas the two flexible branches of the dendron are located outside the active site (Fig. 4). To some extent, one branch reaches in the major groove of the primer/template duplex, indicated by some electron density in this region. The zoom into the active site shows that the finger domain by the O helix packs against the nascent base pair, resulting in a complex poised for catalysis (Fig. S7). Interestingly, in this case the amino acid Arg660 interacts with the phosphate-backbone of the primer 3′-terminus as well as with amide functionality of the propargylamide linkage. Although the described interaction pattern may stabilize Arg660, we observe a high B-factor of the amino acid side chain 97 Å2, indicating that the position of Arg660 is tenuous. The geometric freedom of Arg660 may explain the 137-fold reduced incorporation efficiency of dTdendTP compared to natural dTTP. In addition, the propargylamide linkage is interacting with the phosphate backbone of the primer 3′-terminus via hydrogen-bonding interactions (Fig. 3). Furthermore, we observe a second hydrogen bond formed between the carbonyl group of an amide in the dendron moiety and the nucleobase of the primer 3′-terminus (Fig. 3D). These hydrogen-bonding interactions might support a nucleotide position and conformation better suited for incorporation compared to dTspinTP and more similar to dTTP.

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Structure of KlenTaq in complex with dTdendTP (KlenTaqdend). (A) The close-up view of KlenTaqdend (orange) shows the incoming dTdendTP (purple), the O helix with amino acid side chain R660, the primer and template strand. The interaction network is indicated in dashed lines as well as the Watson–Crick base pairing interaction between dTdendTP and adenine of the template strand. R660 is orientated to allow interaction with the primer 3′-terminus. The amine group of the propargyl-amine-linkage shows an additional interaction to the primer 3′-terminus. (B) Superimposition of KlenTaqdend and KlenTaq (green, PDB: 1QTM) highlights the different orientation of the amino acid side chain R660. (C) The interaction pattern of the phosphate backbone at the primer 3′-terminus with R660 and the propargylamide linkage is shown in dashed lines. The distance of the α-phosphate to the primer 3′-terminus is 3.7 Å (highlighted in dashed lines). (D) Interaction network of dTdendTP with the primer 3′-terminus toward the phosphate backbone and the nucleobase is indicated with dashed lines. The final refined model map 2mFo-DFc at 1σ is shown for dTdendTP.

Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

Structure of KlenTaqdend ternary complex. (A) The overall structure of KlenTaqdend and the Connolly surface is shown. The incoming dTdendTP is highlighted in sticks and indicated with a red arrow. (B) The close-up view of the incoming dTdendTP illustrates that the dendrimer structure is placed in a cleft between the protein and primer/template duplex.

Discussion

Hitherto, structural data of DNA polymerases processing C5 modified pyrimidines that could serve as a guide for structure design of suitable nucleotide modifications was lacking. We present two crystal structures of KlenTaq processing differently modified nucleotide analogs. The protein structures in KlenTaqspin and KlenTaqdend exhibit the incoming nucleotide in a position poised for catalysis similar to the structures reported for KlenTaq bound to primer/template and a nucleotide with natural nucleobase (26, 28). Only subtle differences in terms of alignment of the incoming nucleotides, magnesium ions, and water molecules are observed (Fig. S7). This suggests that the enzyme follows similar mechanisms to promote catalysis. However, the structures mainly differ in the position of amino acid side chain Arg660. In KlenTaqspin the modified dTspinTP obviates Arg660 to undergo stabilizing interactions with the primer. In contrast, hydrogen-bonding interactions of the enzyme with dTdendTP via the amide functionality of the propargylamide linker are observed in KlenTaqdend. The propargylamide linkage as present in dTdendTP offers the opportunity to stabilize the closed complex poised for catalysis, which may have beneficial effects on the incorporation efficiency. Amino acid sequence alignment of several DNA polymerases that belong to the same sequence family as KlenTaq (i.e., A-family) discloses that the respective arginine is within motif B (32) and is conserved in bacteria (Fig. S8). Thus, it is likely that the depicted mechanism to stabilize the enzyme/substrate complex applies to other DNA polymerases in this sequence family as well.

The results suggest that the modifications are better tolerated by a DNA polymerase if they are attached via linkers that have hydrogen-bonding capability with the enzyme at the depicted positions. In fact, several examples of dNTPs that are processed by DNA polymerases and modified via a C5 propargylamide linker have been reported (1–3, 5, 6, 8, 10–14, 16, 17, 21, 23). These insights suggest that implementing of functionalities with hydrogen binding capability at the discussed position in modified dNTPs may improve their substrate properties. These design guidelines for the development of new modified dNTPs, in combination with directed evolution of DNA polymerases (33–35), will spur the development of future applications.

Materials and Methods

Adducted Protein, Nucleotides, and Oligonucleotides.

Protein expression and purification were conducted as described (25–28, 30, 31). Modified thymidine-5′-triphosphates dTRTPs were synthesized as described previously. Unmodified dNTPs are commercially available (Roche).

DNA oligonucleotides for enzyme kinetics were synthesized on an Applied-Biosystems 392 DNA/RNA synthesizer and purified by reversed phase HPLC (DMT-ON) and afterwards by preparative PAGE on a 12% polyacrylamide gel containing 8 M urea (DMT-OFF). DNA primer oligonucleotides were labeled with [γ-32P] ATP using T4 polynucleotide kinase (Fermentas) according to the procedure recommended by the manufacturer.

Single Nucleotide Incorporation Assay.

Incorporation opposite dA: 15 μl of the KlenTaq reactions contained 50 nM primer (5'-GTG GTG CGA AAT TTC TGA CAG ACA), 75 nM template (5′-GTG CGT CTG TCA TGT CTG TCA GAA ATT TCG CAC CAC), 200 μM dNTPs in buffer (20 mM Tris HCl pH 7.5, 50 mM NaCl, and 2 mM MgCl2) and 200 nM of KlenTaq polymerase. Reaction mixtures were incubated at 37 °C. Incubation times are provided in the respective figure legends. Primer was labeled using [γ-32P]-ATP according to standard techniques. Reactions were stopped by addition of 45 μl stop solution (80% [v/v] formamide, 20 mM EDTA, 0.25% [w/v] bromophenol blue, 0.25% [w/v] xylene cyanol) and analyzed by 12% denaturing PAGE. Visualization was performed by phosphoimaging.

Pre-Steady-State Enzyme Kinetics.

The rate of single turnover, single nucleotide incorporation was determined using rapid quench flow kinetics on a chemical quench flow apparatus (RQF-3, KinTek Corp.) as previously described (36). For reaction times longer than 5 s manual quenching was performed. In brief, 15 μl of radiolabelled primer/template complex (50 nM) and DNA polymerase (500 nM) in reaction buffer (RQF buffer, Tris HCl pH 7.5 20 mM, NaCl 50 mM, MgCl2 2 mM) (29) were rapidly mixed with 15 μl of a dNTP solution in reaction buffer at 37 °C. Quenching was achieved by 0.3M EDTA solution (pH 7.0) at defined time intervals. For the investigation of dTRTP incorporation a 24 nt primer (5'-GTG GTG CGA AAT TTC TGA CAG ACA) and a 36 nt template (5′-GTG CGT CTG TCA TGT CTG TCA GAA ATT TCG CAC CAC) were applied. Quenched samples were analyzed on a 12% denaturing PAGE followed by phosphor imaging. For kinetic analysis experimental data were fit by nonlinear regression using the program GraphPad Prism 4. The data were fit to a single exponential equation: [conversion] = A∗(1- exp(-kobst)). The observed catalytic rates (kobs) were then plotted against the dNTP concentration used and the data were fitted to a hyperbolic equation Embedded Image to determine the apparent KD and kcat of the incoming nucleotide. The incorporation efficiency is given by kpol/KD. The depicted data derived from double repeated experiments.

Crystallization and Structure Determination.

The closed ternary complexes of KlenTaq were obtained by incubating KlenTaq in presence of DNA primer (5′-d(GAC CAC GGC GC)-3′), template (5′-d(AAA AGG CGC CGT GGT C)-3′), ddCTP and one of the 5-modified dTRTP. Thereby, one ddCMP is incorporated, while the corresponding 5-modified dTRTP is captured in the position waiting for insertion.

KlenTaqspin: The crystallization was set up using purified KlenTaq (11 mg/ml; buffer: 20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol), DNA template/primer duplex, ddCTP and dTspinTP in a molar ratio of 1∶3∶10∶20 and in presence of 20 mM MgCl2. The crystallization solution was mixed in 1∶1 ratio with the reservoir solution 0.05 M Tris HCl, pH 9, 0.2 M NH4Cl, 0.01 M CaCl2, and 26% PEG 4000.

KlenTaqdend: The crystallization was set up using purified KlenTaq (11 mg/ml; buffer: 20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol), DNA template/primer duplex, ddCTP and dTdendTP in a molar ratio of 1∶3∶10∶20 and in presence of 20 mM MgCl2. The crystallization solution was mixed in 1∶1 ratio with the reservoir solution containing 0.05 M sodium cacodylate (pH 6.5), 0.2 M NH4OAc, 0.01 M Mg(OAc)2, and 26% PEG 8000.

Crystals were produced by the hanging drop vapor diffusion method, equilibrating against 1 ml of the reservoir solution for 5 d at 18 °C. They were frozen in liquid nitrogen and kept at 100 K during data collection. Data were measured at the beamline PXIII (X06DA) at the Swiss Light Source of the Paul Scherrer Institute (PSI) in Villigen, Switzerland, at a wavelength 1.000 Å and using a Mar225 CCD detector. Data reduction was performed with the XDS package (37, 38). The structures were solved by difference Fourier techniques using KlenTaq wild type (PDB 1QTM) as model. Refinement was performed with PHENIX (39) and model rebuilding was done with COOT (40). The structures were refined to a resolution of 2.0 Å in KlenTaqspin and 1.9 Å in KlenTaqdend. Both structures were in the same space group P3121, with cell dimensions a, b 109.0 Å, c 91.5 Å for KlenTaqspin, and a, b 107.8 Å, c 90.2 Å for KlenTaqdend, respectively. Figures were made with PyMOL (41). The Ramachandran statistics for KlenTaqspin show that 91.5% of the residues are in the most favored regions, 7.9% in additional allowed regions, 0.4% in generously allowed and 0.2% in disallowed regions as defined PROCHECK (42). For KlenTaqdend the values are 92.3%, 7.5%, 0%, and 0.2%, respectively.

Acknowledgments

This work was partly funded by the German Excellence Initiative.

Footnotes

  • 1To whom correspondence should be addressed: E-mail: andreas.marx{at}uni-konstanz.de.
  • Author contributions: S.O. and A.M. designed research; S.O. performed research; S.O. and A.B. contributed new reagents/analytic tools; S.O., W.W., K.D., and A.M. analyzed data; and S.O., K.D., and A.M. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3OJU and 3OJS).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1013804107/-/DCSupplemental.

References

  1. ↵
    1. Bentley DR,
    2. et al.
    (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53–59.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Bowers J,
    2. et al.
    (2009) Virtual terminator nucleotides for next-generation DNA sequencing. Nat Methods 6:593–595.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Ruparel H,
    2. et al.
    (2005) Design and synthesis of a 3′-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis. Proc Natl Acad Sci USA 102:5932–5937.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Harris TD,
    2. et al.
    (2008) Single-molecule DNA sequencing of a viral genome. Science 320:106–109.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Guo J,
    2. et al.
    (2008) Four-color DNA sequencing with 3-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc Natl Acad Sci USA 105:9145–9150.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Pollack JR,
    2. et al.
    (1999) Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 23:41–46.
    OpenUrlPubMed
  7. ↵
    1. Perou CM,
    2. et al.
    (2000) Molecular portraits of human breast tumours. Nature 406:747–752.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Seo TS,
    2. et al.
    (2005) Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides. Proc Natl Acad Sci USA 102:5926–5931.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Weisbrod SH,
    2. Marx A
    (2007) A nucleoside triphosphate for site-specific labelling of DNA by the Staudinger ligation. Chem Commun 18:1828–1830.
    OpenUrl
  10. ↵
    1. Mayer G
    (2009) The chemical biology of aptamers. Angew Chem Int Edit 48:2672–2689.
    OpenUrlCrossRef
  11. ↵
    1. Thum O,
    2. Jäger S,
    3. Famulok M
    (2001) Functionalized DNA: A new replicable biopolymer. Angew Chem Int Edit 40:3990–3993.
    OpenUrlCrossRef
  12. ↵
    1. Weizman H,
    2. Tor Y
    (2002) Redox-active metal-containing nucleotides: Synthesis, tunability, and enzymatic incorporation into DNA. J Am Chem Soc 124:1568–1569.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Giller G,
    2. et al.
    (2003) Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2′-deoxyribonucleoside-5′-triphosphates. Nucleic Acids Res 31:2630–2635.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Jäger S,
    2. Famulok M
    (2004) Generation and enzymatic amplification of high-density functionalized DNA double strands. Angew Chem Int Edit 43:3337–3340.
    OpenUrlCrossRef
  15. ↵
    1. Anderson JP,
    2. Angerer B,
    3. Loeb LA
    (2005) Incorporation of reporter-labeled nucleotides by DNA polymerases. Biotechniques 38:257–264.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Ohbayashi T,
    2. et al.
    (2005) Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase. Org Biomol Chem 3:2463–2468.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Jäger S,
    2. et al.
    (2005) A versatile toolbox for variable DNA functionalization at high density. J Am Chem Soc 127:15071–15082.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Srivatsan SG,
    2. Tor Y
    (2007) Fluorescent pyrimidine ribonucleotide: Synthesis, enzymatic incorporation, and utilization. J Am Chem Soc 129:2044–2053.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Čapek P,
    2. et al.
    (2007) An efficient method for the construction of functionalized DNA bearing amino acid groups through cross-coupling reactions of nucleoside triphosphates followed by primer extension or PCR. Chem-Eur J 13:6196–6203.
    OpenUrl
  20. ↵
    1. Gramlich PME,
    2. Wirges CT,
    3. Manetto A,
    4. Carell T
    (2008) Postsynthetic DNA modification through the copper-catalyzed azide-alkyne cycloaddition reaction. Angew Chem Int Edit 47:8350–8358.
    OpenUrlCrossRef
  21. ↵
    1. Hocek M,
    2. Fojta M
    (2008) Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids. Org Biomol Chem 6:2233–2241.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Obeid S,
    2. Yulikow M,
    3. Jeschke G,
    4. Marx A
    (2008) Enzymatic synthesis of multi spin-labeled DNA. Angew Chem Int Edit 47:6782–6785.
    OpenUrlCrossRef
  23. ↵
    1. Baccaro A,
    2. Marx A
    (2010) Enzymatic synthesis of organic polymer-grafted DNA. Chem-Eur J 16:218–226.
    OpenUrl
  24. ↵
    1. Korolev S,
    2. Nayal M,
    3. Barnes WM,
    4. Di Cera E,
    5. Waksman G
    (1995) Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: Structural basis for thermostability. Proc Natl Acad Sci USA 92:9264–9268.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Li Y,
    2. Korolev S,
    3. Waksman G
    (1998) Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J 17:7514–7525.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Li Y,
    2. Kong Y,
    3. Korolev S,
    4. Waksman G
    (1998) Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates. Protein 7:1116–1123.
    OpenUrlCrossRef
  27. ↵
    1. Li Y,
    2. Mitaxov V,
    3. Waksman G
    (1999) Structure-based designof Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. Proc Natl Acad Sci USA 96:9491–9496.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Li Y,
    2. Waksman G
    (2001) Crystal structures of a ddATP-, ddTTP-, ddCTP-, and ddGTP-trapped ternary complex of Klentaq1: Insights into nucleotide incorporation and selectivity. Protein Sci 10:1225–1233.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Rothwell PJ,
    2. Mitaksov V,
    3. Waksman G
    (2005) Motions of the fingers subdomain of klentaq1 are fast and not rate limiting: Implications for the molecular basis of fidelity in DNA polymerases. Mol Cell 19:345–355.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Obeid S,
    2. et al.
    (2010) Replication through an abasic DNA lesion: Structural basis for adenine selectivity. EMBO J 29:1738–1747.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Betz K,
    2. et al.
    (2010) Structures of DNA polymerases caught processing size-augmented nucleotide probes. Angew Chem Int Edit 49:5181–5184.
    OpenUrlCrossRef
  32. ↵
    1. Delarue M,
    2. Poch O,
    3. Tordo N,
    4. Moras D,
    5. Argos P
    (1990) An attempt to unify the structure of polymerases. Protein Eng 3:461–467.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Ramsay N,
    2. et al.
    (2010) CyDNA: Synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase. J Am Chem Soc 132:5096–5104.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Leconte AM,
    2. et al.
    (2010) Directed evolution of DNA polymerases for next-generation sequencing. Angew Chem Int Ed 49:5921–5924.
    OpenUrlPubMed
  35. ↵
    1. Staiger N,
    2. Marx A
    (2010) A DNA polymerase with increased reactivity for ribonucleotides and C5-modified deoxyribonucleotides. ChemBioChem 11:1963–1966.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Di Pasquale F,
    2. et al.
    (2008) Opposed steric constraints in human DNA polymerase beta and E. coli DNA polymerase I. J Am Chem Soc 130:10748–10757.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Kabsch W
    (2010) XDS. Acta Crystallogr D 66:125–132.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Kabsch W
    (2010) Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D 66:133–144.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Adams PD,
    2. et al.
    (2002) PHENIX: Building new software for automated crystallographic structure determination. Acta Crystallogr D 58:1948–1954.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Emsley P,
    2. Cowtan K
    (2004) Coot: Model-building tools for molecular graphics. Acta Crystallogr D 60:2126–2132.
    OpenUrlCrossRefPubMed
  41. ↵
    1. DeLano WL
    (2002) The PyMOL Molecular Graphics System (DeLano Scientific, Palo Alto, CA).
  42. ↵
    1. Laskowski RA,
    2. MacArthur MW,
    3. Moss DS,
    4. Thornton JM
    (1993) PROCHECK—a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291.
    OpenUrlCrossRef
PreviousNext
Back to top
Article Alerts
Email Article

Thank you for your interest in spreading the word on PNAS.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase
(Your Name) has sent you a message from PNAS
(Your Name) thought you would like to see the PNAS web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase
Samra Obeid, Anna Baccaro, Wolfram Welte, Kay Diederichs, Andreas Marx
Proceedings of the National Academy of Sciences Dec 2010, 107 (50) 21327-21331; DOI: 10.1073/pnas.1013804107

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase
Samra Obeid, Anna Baccaro, Wolfram Welte, Kay Diederichs, Andreas Marx
Proceedings of the National Academy of Sciences Dec 2010, 107 (50) 21327-21331; DOI: 10.1073/pnas.1013804107
Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Mendeley logo Mendeley
Proceedings of the National Academy of Sciences: 107 (50)
Table of Contents

Submit

Sign up for Article Alerts

Article Classifications

  • Physical Sciences
  • Chemistry
  • Biological Sciences
  • Biophysics and Computational Biology

Jump to section

  • Article
    • Abstract
    • Results
    • Discussion
    • Materials and Methods
    • Acknowledgments
    • Footnotes
    • References
  • Figures & SI
  • Info & Metrics
  • PDF

You May Also be Interested in

Surgeons hands during surgery
Inner Workings: Advances in infectious disease treatment promise to expand the pool of donor organs
Despite myriad challenges, clinicians see room for progress.
Image credit: Shutterstock/David Tadevosian.
Setting sun over a sun-baked dirt landscape
Core Concept: Popular integrated assessment climate policy models have key caveats
Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
Double helix
Journal Club: Noncoding DNA shown to underlie function, cause limb malformations
Using CRISPR, researchers showed that a region some used to label “junk DNA” has a major role in a rare genetic disorder.
Image credit: Nathan Devery.
Steamboat Geyser eruption.
Eruption of Steamboat Geyser
Mara Reed and Michael Manga explore why Yellowstone's Steamboat Geyser resumed erupting in 2018.
Listen
Past PodcastsSubscribe
Multi-color molecular model
Enzymatic breakdown of PET plastic
A study demonstrates how two enzymes—MHETase and PETase—work synergistically to depolymerize the plastic pollutant PET.
Image credit: Aaron McGeehan (artist).

Similar Articles

Site Logo
Powered by HighWire
  • Submit Manuscript
  • Twitter
  • Facebook
  • RSS Feeds
  • Email Alerts

Articles

  • Current Issue
  • Special Feature Articles – Most Recent
  • List of Issues

PNAS Portals

  • Anthropology
  • Chemistry
  • Classics
  • Front Matter
  • Physics
  • Sustainability Science
  • Teaching Resources

Information

  • Authors
  • Editorial Board
  • Reviewers
  • Librarians
  • Press
  • Site Map
  • PNAS Updates

Feedback    Privacy/Legal

Copyright © 2021 National Academy of Sciences. Online ISSN 1091-6490