Structural Insights into the Processing of Nucleobase-Modified

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Structural Insights into the Processing of Nucleobase-Modified Nucleotides by DNA Polymerases Audrey Hottin and Andreas Marx* Department of Chemistry and Konstanz Research School Chemical Biology University of Konstanz Universitätsstrasse 10, 78457 Konstanz, Germany CONSPECTUS: The DNA polymerase-catalyzed incorporation of modified nucleotides is employed in many biological technologies of prime importance, such as next-generation sequencing, nucleic acid-based diagnostics, transcription analysis, and aptamer selection by systematic enrichment of ligands by exponential amplification (SELEX). Recent studies have shown that 2′-deoxynucleoside triphosphates (dNTPs) that are functionalized with modifications at the nucleobase such as dyes, affinity tags, spin and redox labels, or even oligonucleotides are substrates for DNA polymerases, even if modifications of high steric demand are used. The position at which the modification is introduced in the nucleotide has been identified as crucial for retaining substrate activity for DNA polymerases. Modifications are usually attached at the C5 position of pyrimidines and the C7 position of 7-deazapurines. Furthermore, it has been shown that the nature of the modification may impact the efficiency of incorporation of a modified nucleotide into the nascent DNA strand by a DNA polymerase. This Account places functional data obtained in studies of the incorporation of modified nucleotides by DNA polymerases in the context of recently obtained structural data. Crystal structure analysis of a Thermus aquaticus (Taq) DNA polymerase variant (namely, KlenTaq DNA polymerase) in ternary complex with primer-template DNA and several modified nucleotides provided the first structural insights into how nucleobase-modified triphosphates are tolerated. We found that bulky modifications are processed by KlenTaq DNA polymerase as a result of cavities in the protein that enable the modification to extend outside the active site. In addition, we found that the enzyme is able to adapt to different modifications in a flexible manner and adopts different amino acid side-chain conformations at the active site depending on the nature of the nucleotide modification. Different “strategies” (i.e., hydrogen bonding, cation−π interactions) enable the protein to stabilize the respective protein−substrate complex without significantly changing the overall structure of the complex. Interestingly, it was also discovered that a modified nucleotide may be more efficiently processed by KlenTaq DNA polymerase when the 3′-primer terminus is also a modified nucleotide instead of a nonmodified natural one. Indeed, the modifications of two modified nucleotides at adjacent positions can interact with each other (i.e., by π−π interactions) and thereby stabilize the enzyme−substrate complex, resulting in more efficient transformation. Several studies have indicated that archeal DNA polymerases belonging to sequence family B are better suited for the incorporation of nucleobase-modified nucleotides than enzymes from family A. However, significantly less structural data are available for family B DNA polymerases. In order to gain insights into the preference for modified substrates by members of family B, we succeeded in obtaining binary structures of 9°N and KOD DNA polymerases bound to primer-template DNA. We found that the major groove of the archeal family B DNA polymerases is better accessible than in family A DNA polymerases. This might explain the observed superiority of family B DNA polymerases in polymerizing nucleotides that bear bulky modifications located in the major groove, such as modification at C5 of pyrimidines and C7 of 7-deazapurines. Overall, this Account summarizes our recent findings providing structural insight into the mechanism by which modified nucleotides are processed by DNA polymerases. It provides guidelines for the design of modified nucleotides, thus supporting future efforts based on the acceptance of modified nucleotides by DNA polymerases.

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(SELEX).4 Recent advances in the field, such as the SOMAmerbased proteomics platform, enable large-scale protein identifications providing new disease-specific biomarkers that are essential for developing new diagnostics and treatments.5 Obviously, DNA polymerases have not been “evolved” by nature to tolerate non-natural nucleotides, and thus, it is enigmatic how they are able to do so.

INTRODUCTION For many key biotechnological applications, the capability of DNA polymerases to accept modified 2′-deoxynucleoside triphosphates (dNTPs) is essential. These include nextgeneration and single-molecule sequencing approaches1,2 that have revolutionized the field of molecular genetics. The sequencing of complete genomes is now possible, thus helping the detection of genetic alterations that lead to severe diseases.3 Another innovative technology that employs chemically modified nucleotides is the in vitro selection of aptamers by systematic enrichment of ligands by exponential amplification © XXXX American Chemical Society

Received: December 16, 2015

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DOI: 10.1021/acs.accounts.5b00544 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. Structure of 2′-deoxynucleoside triphosphates and the Watson−Crick base pairs. The dashed lines indicate hydrogen bonds.

Figure 2. Structures of labeled nucleotides successfully incorporated into DNA by DNA polymerases. T5 = 5-substitued 2′-deoxyuridine-5′triphosphate. B = 2′-deoxyribonucleoside triphosphate modified at C5 of pyrimidines or C7 of 7-deazapurines.

8-bromo- and 8-methyl-dATP are suitable substrates for DNA polymerase while the respective 8-phenyl modification is too bulky to be incorporated.17 Perrin and co-workers reported the successful use of C8-imidazole-modified 2′-deoxyadenosine in PCR reactions.18 To favor the acceptance of unnatural substrates, the DNA polymerase used for catalysis has to be carefully chosen. For instance, Famulok and co-workers reported the preparation of highly functionalized oligodeoxynucleotides (ODNs) and discovered that the use of DNA polymerases from sequence family B is superior to the use of family A DNA polymerases.10 The same observation was also made by Sawai and co-workers for a broad range of C5-modified pyrimidine nucleoside triphosphates.12,19−21 The incorporation efficiency of unnatural substrates also depends on the linker used to anchor the modification to the nucleobase, especially if sterically demanding groups are attached.12 Bulky labels, such as fluorescent dyes (i.e., cyanines, fluorescein, rhodamine, Bodipy) or affinity tags (biotin) are well-accepted, when being attached to the nucleobase via a long flexible linker (Figure 2).12,22−24 This strategy has been wellemployed in next-generation sequencing approaches for the design of fluorescently labeled reversible terminator nucleotides.1,25,26 The ability to incorporate highly sterically demanding modifications raises the question of size limits of

Reports on studies of DNA polymerases and their interplay with modified nucleotides strongly indicate that both the position at which the modification is introduced and the kind of modification play essential roles in the acceptance of a modified nucleotide by a DNA polymerase. In principle, modifications can be introduced in the sugar moiety, the phosphate, and the nucleobase. While nucleobase modifications are processed most efficiently, modifications of the sugar and phosphate are poorly tolerated.6 In principle, suitable positions for the attachment of modifications are position 7 or 8 of purines and position 5 or 6 of pyrimidines because they do not affect the Watson−Crick base pairing and are well-accommodated in the major groove of the DNA duplex (Figure 1). Recent investigations indicate that enzymatic synthesis of base-modified DNA is almost exclusively performed by pyrimidines modified at C5 and 7-deazapurines modified at C7.7−12 This is mainly due to the excellent substrate properties shown by these analogues not only in primer extension (PEX) but also in the polymerase chain reaction (PCR). New perspectives arise from the fact that certain dNTPs modified at these positions are even enhanced substrates for incorporation compared with the natural dNTPs.13,14 In contrast, nucleotides modified at C6 of pyrimidines or C8 of purines are less favored for incorporation by DNA polymerases.10,11,15,16 Hocek and co-workers showed that small substituents such as B


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Figure 3. Chemical structures of the modified nucleotides dN*TP, dN**TP, dNarylTP, dTdendTP, and dTspinTP.

Figure 4. Strategy for the crystallization of KlenTaq DNA polymerase bound to primer/template and modified nucleotide in the catalytic site.

base modifications tolerated by DNA polymerases. To address this question, we aimed at synthesizing polymer-grafted DNA by enzymatic incorporation. We successfully prepared DNA strands functionalized with polymer blocks such as poly(ethylene glycol) monomethyl ethers or branched polyamido dendrons by primer extension (PEX) using 9°N DNA polymerase.27 More recently, we found that ODNs are wellsuited modifications of nucleotides for incorporation by DNA polymerases.28 Given the steric demand of ODN strands, we were intrigued to observe that a variant of Thermus aquaticus (Taq) DNA polymerase I (KlenTaq DNA polymerase) and Therminator DNA polymerase catalyze the incorporation of ODN-modified nucleotides even when ODN strands with up to 40 nucleotides were employed as “cargo”. Furthermore, we found that nucleotides modified with a structured Gquadruplex-derived DNAzyme are also substrates for KlenTaq and Klenow fragment exo− DNA polymerases.29 These examples prove the startling ability of DNA polymerases to process 5-substituted pyrimidine and 7substituted 7-deazapurine nucleoside triphosphates even though the modifications are very bulky. This Account describes our recent efforts to understand the mechanism by which DNA polymerases accept such bulky modifications through the use of structural data. We discuss the incorporation efficiencies of modified nucleotides as well as the proficiencies

of two DNA polymerase families (A vs B) in processing nucleotides on the basis of our recently obtained structural data.

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HOW ARE HEAVILY MODIFIED NUCLEOTIDES PROCESSED BY DNA POLYMERASES? To explore the mechanism of acceptance of modified nucleotides, we crystallized DNA polymerases in ternary complex with DNA primer/template duplex and bound to a modified nucleotide substrate placed in the active site. We chose KlenTaq DNA polymerase, taking advantage of the comprehensive characterization of the enzyme on a structural level, in particular with available respective ternary structures of the enzyme in complex with the four incoming nucleotide triphosphates.30,31 As artificial substrates, we employed a series of four modified 2′-deoxynucleoside-5′-triphosphates (dN*TPs) carrying aminopentynyl modifications linked either at C5 of pyrimidines or at C7 of 7-deazapurines (Figure 3). The crystals were obtained by a strategy previously used for KlenTaq DNA polymerase bound to unmodified substrates (Figure 4).31−34 The primer contained a ddC at the 3′-end to capture the incoming modified dNTP at the active site. Because of the lack of a 3′-OH at the primer terminus, incorporation of the modified nucleotide is excluded. The structures were solved by difference Fourier techniques and provide snapshots of C


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Figure 5. Structures of KlenTaq DNA polymerase bound to dN*TPs. The dashed lines highlight the Watson−Crick base-pairing interactions and the distance of the α-phosphate to the primer. All distances are in Å. (A−D) Close-up views showing KlenTaq DNA polymerase in complex with (A) dT*TP (magenta, PDB ID 4DFJ), (B) dC*TP (pink, 4DFM), (C) dA*TP (dark blue, 4DF8), (D) dG*TP (blue, 4DFP) and overlays with KlenTaq(ddTTP) (1QTM), KlenTaq(ddCTP) (3KTQ), KlenTaq(ddATP) (1QSY), and KlenTaq(ddGTP) (1QSS), respectively (gray). (E) Closeup view of the modification of dT**TP (magenta, 4DFK) pointing outside the protein. The finger, thumb, and palm domains are depicted in pale blue, green, and orange, respectively. (F) Same as (E) with dA**TP (dark blue, 4DF4).

modified triphosphates bound to KlenTaq DNA polymerase prior to incorporation at high resolution (1.9−2.1 Å). The basic fold of a DNA polymerase is generally compared to the shape of a right hand in which the finger, palm, and thumb subdomains form the DNA-binding crevice, and this was found by us as well.35 In KlenTaq DNA polymerase, the finger domain changes its conformation from an open state to a closed state by reorientation of the O-helix in order to generate an active enzyme−substrate complex.33 In our studies, each structure of KlenTaq DNA polymerase bound to a modified dN*TP and the primer/template duplex forms a closed ternary complex. The O-helix packs against the nascent base pair, thereby closing the crevice (Figure 5A−D). The structures adopt an overall conformation very similar to the cases in which unmodified substrates were employed (rootmean-square deviation (RMSD) for Cα = 0.33−0.38 Å).30,33

The complexes are trapped in a state prior to catalysis. As observed in the cases in which unmodified substrates were used, two catalytic metal ions are octahedrally coordinated by the phosphate groups of dN*TP, three carboxylates (Asp610, Asp785, and Tyr611) and two water molecules. All of the nucleobases show the usual binding interactions, such as Watson−Crick base pairing to the templating base or stacking interactions with the primer 3′-terminal base. In comparison with the cases in which unmodified substrates were used, Arg660 is displaced as a result of steric hindrance caused by the modification. The reorientation of Arg660 illustrates the plasticity of DNA polymerases to adapt to the structure of the incoming nucleotide. To extend our study to longer modifications, we further crystallized KlenTaq DNA polymerase bound to thymidine or adenosine triphosphates bearing a (hydroxydecanoyl)D


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Figure 6. Structures of KlenTaq DNA polymerase in complex with dNxTPs. The dashed lines highlight the interaction patterns. All distances are in Å. (A) Close-up view showing dTspinTP bound to KlenTaq DNA polymerase (brown, PDB ID 3OJU) superimposed with KlenTaq(1QTM) (gray). (B, C) Same as (A) for the structure containing dTarylTP (gold, 4ELT) and dTdendTP (green, 3OJS), respectively. (D−F) Same complexes as in (A−C), respectively, showing further interaction patterns.

nucleotides’ relative concentration required in order to achieve a 1:1 ratio of incorporation of the natural and modified nucleotides. For example, a ratio of 1:10 reflects that a 10-fold concentration of the modified nucleotide relative to the natural nucleotide is required in order to achieve a 1:1 incorporation ratio. We first investigated the effect of a rigid nonpolar modification by crystallization of KlenTaq DNA polymerase in complex with the spin-labeled nucleotide dTspinTP following the strategy described above (Figure 4).36,37 dTspinTP has a low incorporation efficiency relative to natural dTTP (1:2500), but the overall conformation of KlenTaq(dTspinTP) is very similar to that of KlenTaq(ddTTP).30 dTspinTP is recognized by the enzyme via the usual Watson−Crick base pairing and πstacking interactions with the primer. In analogy to KlenTaq(ddTTP), the O-helix packs against the nascent base pair, and catalytic residues are properly arranged. The most significant difference between the two structures derives from Arg660, which is positioned in a way that makes room for the nitroxide modification (Figure 6A,D). This reorientation prevents the interaction of Arg660 with the primer 3′-end, which is thought to act as a clamp between the finger domain and the DNA duplex.31,32 As a result, the closed conformation is not stabilized by this interaction, which may be responsible for the loss in incorporation efficiency of dTspinTP compared with dTTP. Rigid nonpolar modifications were further analyzed by the introduction of an aromatic ring connected to the nucleobase via an alkyne. For this purpose, we solved crystal structures of KlenTaq DNA polymerase in complex with C5-aryl-modified cytidine and thymidine triphosphate analogues (dCarylTP and dTarylTP, respectively) bound to the active site (Figure 6B,E).38 The two structures have very similar features. All of the amino acids required for catalysis are arranged as in KlenTaq(ddTTP)

aminopentynyl modification as closed ternary complexes (dT**TP and dA**TP, respectively).14 The crystal structures reveal how the modification extends outside the protein, taking different orientations (Figure 5E, F). In dT**TP, the C5 position orients the modification above the O-helix through the cavity mainly formed by the Arg587 side chain and residues from the O-helix (Leu657, Met658, Arg660, Ala661, and Thr664). In the structure containing dA**TP, the C7 position orients the modification above the 5′-triphosphate group through the cleft formed by residues from the palm domain (Asp610, Glu820, and Lys831), amino acid side chains from the O-helix (Arg659, Arg660, and Lys663), and Arg587. The direct comparison of the KlenTaq(dT**TP) and KlenTaq(dA**TP) structures highlights the displacement of mainly two residues, Arg660 and Arg587. The option of a modification to extend to the outside of the protein through the described cavities enables the enzymatic incorporation of even bulky groups into DNA, provided that a long linker is used.

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WHY IS ONE SUBSTRATE BETTER ACCEPTED THAN ANOTHER? On the basis of structural studies, we aimed at gaining insight into the properties that make one modified nucleotide a better substrate than another. Therefore, we first investigated the enzyme’s efficiency at incorporating modified nucleotides in comparison with their natural counterparts. We performed single-nucleotide incorporation experiments in which a respective modified nucleotide directly competes for incorporation with its natural counterpart. The ratio of unmodified versus modified nucleotide incorporation is easily accessible via denaturating polyacrylamide gel electrophoresis (PAGE) analysis and phosphorimaging because of the significantly different retention times resulting from the modification. In the following, the relative incorporation efficiency is shown as the E


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Figure 7. Structures of KlenTaq DNA polymerase in complex with dN**TPs. The dashed lines highlight the interaction patterns. All distances are in Å. (A) Close-up view showing dA**TP bound to KlenTaq DNA polymerase (blue, PDB ID 4DF4) superimposed with KlenTaq(ddATP) (gray, 1QSY). (B) Same as (A) for the structure containing dT**TP (pink, 4DFK) superimposed with KlenTaq(ddTTP) (gray, 1QTM). (C) Overlay of dA**TP (blue) and dT**TP (pink).

natural analogue. A more detailed inspection of the structures of KlenTaq DNA polymerase bound to dN**TPs indicates that the amide functionality of the modification is stabilized by amino acids from the O-helix (Figure 7). The amide group of dT**TP interacts with residues Thr664 and Arg660 through hydrogen bonds, while dA**TP is mainly stabilized by Lys663 and most likely by Arg660. We assume that the increased hydrogen-bonding capabilities of the linker contribute to the proficient processing of dN**TP analogues by KlenTaq DNA polymerase, as observed with dTdendTP. In summary, we found that DNA polymerases interact with the modifications and stabilize unnatural conformations that might improve substrate properties. Positively charged amino acids such as arginine and lysine that are located near the active site enable cation−π interactions with an aromatic ring and also undergo hydrogen bonding with functional groups of the modifications. In the design of modified nucleotides to be efficient substrates for DNA polymerases, these interactions should be considered.

except for residues Arg660 and Arg587. As described above for KlenTaq(dTspinTP), Arg660 makes room for the modification by losing its contacts to the primer. However, Arg587 interacts with the phosphate backbone of the 3′-primer terminus and may stabilize the complex. In addition, Arg587 and Lys663 stabilize the incoming nucleotide via cation−π interactions with the aromatic ring. As a result, dTarylTP is efficiently processed by KlenTaq DNA polymerase (1:7 ratio) and clearly shows better substrate properties than the thymidine analogue dTspinTP (1:2500 ratio), which lacks the aromatic ring. The introduction of a phenyl group at the 7-position of 7-deazaadenosine also resulted in improved substrate activity due to increased π−π stacking in the active site.13 In order to investigate the acceptance of flexible bulky modifications by a DNA polymerase, KlenTaq DNA polymerase bound to the branched polyamide dendron-modified triphosphate dTdendTP was crystallized.27,37 The flexible polyamide branches extend outside the active site without affecting the usual binding interactions of the substrate. Amino acids in the active site are oriented in the same way as observed in KlenTaq(ddTTP) (Figure 6C,F). Arg660 is slightly displaced but maintains its interaction with the primer 3′-end and further interacts with the amide functionality of the propargylamide linker. Other hydrogen-bonding interactions of the amide group toward both the primer phosphate backbone and the nucleobase are present. These stabilizing effects may explain the increased incorporation efficiency of dTdendTP (1:137 ratio) compared with dTspinTP. Small flexible modifications were investigated in the study on aminopentynyl-modified nucleotide dN*TPs, as mentioned before. It was shown that analogues bearing a short flexible chain are good substrates for DNA polymerase (i.e., 1:1.1 ratio for dC*TP),14 probably because the chain induces only minor disturbances in the ternary complex. dN**TPs modified with a long chain containing an amide group have even better substrate properties. For instance, dA**TP is incorporated with 2-fold better efficiency than natural dATP, whereas dA*TP is incorporated with 1.5-fold lower efficiency than the

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CONSECUTIVE INCORPORATION OF MODIFIED NUCLEOTIDES The enzymatic preparation of highly functionalized ODNs is crucial for many applications and requires multiple incorporations of modified nucleotides by PEX or PCR.1,2 This has already been demonstrated for a broad range of modifications.12,39−41 However, multiple insertions of modified bases in adjacent positions can be challenging, thereby limiting the scope of applications. To overcome this obstacle, we aimed to gain structural insight into the incorporation of two consecutively modified nucleotides using C5-aryl-modified 2′deoxynucleoside-5′-cytidine triphosphates dCarylTP.42 Competitive PEX experiments using a dCarylTP/dCTP mixture revealed that the extension of a dCaryl-terminated primer is more efficiently processed by KlenTaq DNA polymerase than the extension of a dC-terminated primer, with 7- and 27-fold reductions in the efficiency relative to natural dCTP, respectively. By means of crystal structure studies, we further F


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Figure 8. Structures of KlenTaq DNA polymerase in complex with dNxTPs. The dashed lines highlight the interaction patterns. All distances are in Å. (A) Close-up view of KlenTaq DNA polymerase bound to ddCarylTP and the ddCaryl-terminated primer (purple, PDB ID 4EVL). (B) Same as (A) superimposed with KlenTaq(ddCTP) (gray, 3KTQ). (C) Same as (A) superimposed with KlenTaq(dCarylTP) (gold, 4ELU). (D) Zoom into the active site. Metal ions are depicted as dark-green (Ca2+) or light-green (Mg2+) spheres. (left) Octahedral coordination of metal ion A in KlenTaq(ddCaryl−ddCarylTP). (right) Same as (left) but superimposed with KlenTaq(ddCTP) (gray, 3KTQ) and showing the coordination of metal ion A by five ligands.

Figure 9. Comparison of the DNA environments in family A and B DNA polymerases. (A) KlenTaq DNA polymerase (PDB ID 3SZ2) and (B) KOD DNA polymerase (4K8Z) in binary complex are shown with the domains colored in different shades of blue and the tip of the thumb domain in red. The primer and template backbones are shown in gray and black, respectively, in KlenTaq DNA polymerase and in pink and purple, respectively, in KOD DNA polymerase, with the C5 position of pyrimidine and C7 position of 7-deazapurine nucleobases marked as spheres. (C) Zoom into the major groove at the thumb tip site in (top) KlenTaq DNA polymerase and (bottom) KOD DNA polymerase.

rather similar to the one observed in KlenTaq(ddCTP).33 Furthermore, the active-site arrangement differs from the one in KlenTaq DNA polymerase processing natural substrates. Metal ion B is a Ca2+ ion instead of a Mg2+ ion. In addition, metal ion A is octahedrally coordinated by Asp610, Glu786, and four water molecules, whereas metal ion A in KlenTaq(ddCTP) is coordinated by Asp610, Arg785, two water molecules, and the α-phosphate of the incoming nucleotide. The plasticity of KlenTaq DNA polymerase enables multiple incorporations of modified nucleotides. In particular,

investigated the processing of ddCarylTP by KlenTaq DNA polymerase with a ddCaryl-terminated primer being used. The ternary complex revealed π−π stacking interactions between the two consecutive benzene rings of the modification (Figure 8). As a result, the nucleobase and the phenyl ring are twisted in relation to each other. Arg660 and Arg587 are reoriented in comparison with the previously solved structure of KlenTaq DNA polymerase in complex with one single dCarylTP. The two residues show high flexibility judging from the temperature (B) factors and point to the primer strand in a conformation G


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Accounts of Chemical Research flexible amino acids such as Arg660 and Arg587 near the substrate binding site are able to adapt to the modification.

modifications inside the minor groove. Further relevant differences between the two DNA polymerase families were found in the thumb domain. In both cases, the tip of the thumb domain (residues 506−509 in KlenTaq DNA polymerase and 668−675 in KOD DNA polymerase) interacts with the primer strand, but the contact area differs greatly from one structure to the other. The contact area in the KOD DNA polymerase structure is positioned above the minor groove, interacting with the phosphate backbone, whereas the corresponding area in KlenTaq DNA polymerase extends over the phosphate backbone into the major groove. As a result, modifications at C5 in pyrimidines and C7 in 7-deazapurines located in the major groove might clash with the tip of the domain when processed by KlenTaq DNA polymerase. These results might explain the better efficiency of family B DNA polymerases in incorporating modified nucleotides.

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FAMILY A VERSUS FAMILY B DNA POLYMERASE In several cases, archaeal DNA polymerases belonging to family B (i.e., from Thermococcus sp. 9° N-7 (9°N), Thermococcus kodakarensis (KOD), and Pyrococcus furiosus (Pfu)) appear to be better suited for the incorporation of nucleobase-modified nucleotides than enzymes from family A, such as Taq DNA polymerase.12,19−21,28,39,43−45 The reason for this preferential acceptance of modified substrates has not been elucidated yet, mainly because of the lack of structural data. Although several structures of family A DNA polymerases are available as binary and ternary complexes,14,37,38,42,46 only a few structures for archaeal family B DNA polymerases have been solved to date, mainly as apo structures.47−53 Along these lines,54−57 we investigated the binary structures of two DNA polymerases extensively used for the incorporation of modified substrates, namely, KOD and 9°N DNA polymerases. Family B DNA polymerases are divided into finger, thumb, palm, and N-terminal domains such as in KlenTaq DNA polymerase and have an additional 3′-5′ exonuclease domain (Figure 9). The overall structure in the open state adopts a circular conformation with a central hole, which gets plugged by the finger domain in the closed conformation. Crystal structures were obtained as open binary complexes with a DNA duplex bound in a groove formed by the thumb and palm domains. As no ternary crystal structures have been obtained to date, we discuss the preferential acceptance of modified substrates by comparing the binary structure of KOD DNA polymerase with the binary structure of KlenTaq DNA polymerase (PDB ID 3SZ2) as a representative family A DNA polymerase. We note that the structure of 9°N DNA polymerase is very similar to that of KOD DNA polymerase (i.e., high sequence identity (91%), Cα RMSD = 0.76 Å), thus leading to very similar conclusions. The duplex in the KOD DNA polymerase binary structure adopts a B-form DNA conformation, with most of the 2′deoxyribose moieties showing the ideal puckering for B-DNA (C2′-endo). This is in contrast to the A-form DNA observed near the insertion site in KlenTaq DNA polymerase and also in other family A members such as T7 or Bst DNA polymerases.33,58,59 As B-form DNA is more elongated with a wide-opening major groove compared with A-form DNA, DNA duplex conformation in family B DNA polymerases might favor the acceptance of modified substrates.60 As the KOD and KlenTaq DNA polymerase structures reveal, the majority of direct protein−double-stranded DNA interactions are mediated via the phosphate backbone. While the quantity of these contacts is similar in the two enzymes, significant differences concerning nucleobase or sugar contacts are observed. Six DNA nucleobases interact with five amino acid side chains of KlenTaq DNA polymerase, whereas only five nucleobases interact with three residues of KOD DNA polymerase. In addition, KlenTaq DNA polymerase has another four direct interactions of amino acid side chains with the 2′deoxyribose moiety compared with KOD DNA polymerase. As these sugar and nucleobase contacts are located in the minor groove, one would expect a sterically less hindered minor groove for the family B DNA polymerases. However, in general the DNA minor groove is covered by more protein residues than the major groove irrespective of the DNA polymerase family, thus hindering the further introduction of bulky

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CONCLUSION AND OUTLOOK This Account has highlighted our contributions to the understanding of KlenTaq DNA polymerase in its interplay with artificial substrates. The data obtained to date focus mostly on binding of the modified nucleotide at the catalytic site. However, for proper incorporation into a growing DNA strand, the modification needs to be extended. This process has not been addressed on a structural level more extensively. The acceptance of unnatural substrates depends not only on the nature of the modification and its position in the complex but also on the DNA polymerase. Future investigations in this field should focus on the crystallization of ternary complexes of other DNA polymerases used in biotechnologies (including those from the archaeal family B) bound to the primer/ template complex and to triphosphates (native and unnatural) in the active site. Ternary structures would help to elucidate the mechanisms of fidelity and processivity of those enzymes. With the crystallization strategy used, we can only visualize a snapshot of the situation in the enzyme at a time prior to the reaction. Further studies should evaluate the effects of the modification before or after that state. Time-dependent crystallography should follow processes such as interactions with the finger domain before closure, rearrangements during catalysis, and polymerase reopening.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +49 7531 885139. Fax: +49 7531 885140. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Audrey Hottin obtained her Ph.D. in organic chemistry with Prof J.-B. Behr at the University of Reims in 2013. She is currently a postdoctoral researcher in the group of Andreas Marx. Andreas Marx is Professor of Organic Chemistry and Cellular Chemistry in the Department of Chemistry and Konstanz Research School Chemical Biology of the University of Konstanz.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support by the DFG and support and access to beamlines PXI and PXIII at the Swiss Light Source (SLS) of the Paul Scherrer Institute (Villingen, H


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Switzerland). We thank former and current group members for their contributions to the research highlighted in this Account.

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