Base Pair Geometry Research Articles

Structure of a High Fidelity DNA Polymerase Bound to a Benzo[a]pyrene Adduct That Blocks Replication

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Structure of a purine-purine wobble base pair in the decoding center of the ribosome

Here we report the crystal structures of I.C and I.A wobble base pairs in the context of the ribosomal decoding center, clearly showing that the I.A base pair is of an I(anti).A(anti) conformation, as predicted by Crick. Additionally, the structures enable the observation of changes in the anticodon to allow purine-purine base pairing, the 'widest' base pair geometry allowed in the wobble position.

DNA ligases ensure fidelity by interrogating minor groove contacts.

DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3'-hydroxyl and 5'-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3' side of the ligase junction, but tolerant of mispairs on the 5' side. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3' side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson-Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson-Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson-Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3' side of the ligase junction. The NAD+-dependent ligase, Tth, is more sensitive to the DFT analog on the unligated strand whereas the ATP-dependent T4 ligase is more sensitive to substitutions in the template strand. Electrophoretic gel mobility-shift assays demonstrate that the Tth ligase binds poorly to oligonucleotide substrates containing analogs with altered minor groove contacts.

Trans-hydrogen bond deuterium isotope effects of A:T base pairs in DNA.

The chemical shifts of (13)C2 of adenosine residues of DNA were observed to experience a through-space or trans-hydrogen bond isotope effect as a result of deuterium substitution at the imino hydrogen site of base-paired thymidine residues. NMR measurements of several self-complementary DNA duplexes at natural abundance (13)C in 50% H(2)O, 50% D(2)O solvent mixtures yielded an average trans-hydrogen bond isotope effect, (2h)Delta(13)C2, of -47 ppb. The data suggest that stronger hydrogen bonds have more negative (2h)Delta(13)C2 values, which means that A:T N1.H3 hydrogen bonds increase the anharmonicity of the effective vibrational potential of H3. However, (2h)Delta(13)C2 values do not correlate with intra-residue (2)Delta(13)C4 values of thymidine observed here and earlier (Vakonakis et al., 2003), which suggests that (2h)Delta(13)C2 is not determined entirely by hydrogen bond strength. Instead, the variations observed in (2h)Delta(13)C2 values suggest that they may also be sensitive to base pair geometry.

NMR spectroscopy of RNA.

NMR spectroscopy is a powerful tool for studying proteins and nucleic acids in solution. This is illustrated by the fact that nearly half of all current RNA structures were determined by using NMR techniques. Information about the structure, dynamics, and interactions with other RNA molecules, proteins, ions, and small ligands can be obtained for RNA molecules up to 100 nucleotides. This review provides insight into the resonance assignment methods that are the first and crucial step of all NMR studies, into the determination of base-pair geometry, into the examination of local and global RNA conformation, and into the detection of interaction sites of RNA. Examples of NMR investigations of RNA are given by using several different RNA molecules to illustrate the information content obtainable by NMR spectroscopy and the applicability of NMR techniques to a wide range of biologically interesting RNA molecules.

3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures.

We present a comprehensive software package, 3DNA, for the analysis, reconstruction and visualization of three-dimensional nucleic acid structures. Starting from a coordinate file in Protein Data Bank (PDB) format, 3DNA can handle antiparallel and parallel double helices, single-stranded structures, triplexes, quadruplexes and other complex tertiary folding motifs found in both DNA and RNA structures. The analysis routines identify and categorize all base interactions and classify the double helical character of appropriate base pair steps. The program makes use of a recently recommended reference frame for the description of nucleic acid base pair geometry and a rigorous matrix-based scheme to calculate local conformational parameters and rebuild the structure from these parameters. The rebuilding routines produce rectangular block representations of nucleic acids as well as full atomic models with the sugar-phosphate backbone and publication quality 'standardized' base stacking diagrams. Utilities are provided to locate the base pairs and helical regions in a structure and to reorient structures for effective visualization. Regular helical models based on X-ray diffraction measurements of various repeating sequences can also be generated within the program.

High-fidelity in vivo replication of DNA base shape mimics without Watson-Crick hydrogen bonds.

We report studies testing the importance of Watson-Crick hydrogen bonding, base-pair geometry, and steric effects during DNA replication in living bacterial cells. Nonpolar DNA base shape mimics of thymine and adenine (abbreviated F and Q, respectively) were introduced into Escherichia coli by insertion into a phage genome followed by transfection of the vector into bacteria. Genetic assays showed that these two base mimics were bypassed with moderate to high efficiency in the cells and with very high efficiency under damage-response (SOS induction) conditions. Under both sets of conditions, the T-shape mimic (F) encoded genetic information in the bacteria as if it were thymine, directing incorporation of adenine opposite it with high fidelity. Similarly, the A mimic (Q) directed incorporation of thymine opposite itself with high fidelity. The data establish that Watson-Crick hydrogen bonding is not necessary for high-fidelity replication of a base pair in vivo. The results suggest that recognition of DNA base shape alone serves as the most powerful determinant of fidelity during transfer of genetic information in a living organism.

The annotation of RNA motifs

The recent deluge of new RNA structures, including complete atomic-resolution views of both subunits of the ribosome, has on the one hand literally overwhelmed our individual abilities to comprehend the diversity of RNA structure, and on the other hand presented us with new opportunities for comprehensive use of RNA sequences for comparative genetic, evolutionary and phylogenetic studies. Two concepts are key to understanding RNA structure: hierarchical organization of global structure and isostericity of local interactions. Global structure changes extremely slowly, as it relies on conserved long-range tertiary interactions. Tertiary RNA–RNA and quaternary RNA–protein interactions are mediated by RNA motifs, defined as recurrent and ordered arrays of non-Watson–Crick base-pairs. A single RNA motif comprises a family of sequences, all of which can fold into the same three-dimensional structure and can mediate the same interaction(s). The chemistry and geometry of base pairing constrain the evolution of motifs in such a way that random mutations that occur within motifs are accepted or rejected insofar as they can mediate a similar ordered array of interactions. The steps involved in the analysis and annotation of RNA motifs in 3D structures are: (a) decomposition of each motif into non-Watson–Crick base-pairs; (b) geometric classification of each basepair; (c) identification of isosteric substitutions for each basepair by comparison to isostericity matrices; (d) alignment of homologous sequences using the isostericity matrices to identify corresponding positions in the crystal structure; (e) acceptance or rejection of the null hypothesis that the motif is conserved.

Hybridization ability and base pair geometry of modified deoxycytidine derivatives having a 4-N-carbamoyl group.

Modified oligodeoxyribonucleotides incorporating 4-N-carbamoyldeoxycytidine derivatives were synthesized. The 1H-NMR of 4-N-carbamoyldeoxycytidine suggests that the carbamoyl group forms an intramolecular hydrogen bond with the cytosine ring nitrogen atom and this geometry would inhibit formation of the Watson-Crick base pair with a guanine base. However, the Tm analysis of the modified oligodeoxynucleotides revealed that, in the process of their hybridization with the complementary oligodeoxynucleotides, the orientation of the carbamoyl group changed in a manner where the stable Watson-Crick base pair can be formed with the guanine base.

Motif prediction in ribosomal RNAs Lessons and prospects for automated motif prediction in homologous RNA molecules

The traditional way to infer RNA secondary structure involves an iterative process of alignment and evaluation of covariation statistics between all positions possibly involved in basepairing. Watson–Crick basepairs typically show covariations that score well when examples of two or more possible basepairs occur. This is not necessarily the case for non-Watson–Crick basepairing geometries. For example, for sheared ( trans Hoogsteen/Sugar edge) pairs, one base is highly conserved (always A or mostly A with some C or U), while the other can vary (G or A and sometimes C and U as well). RNA motifs consist of ordered, stacked arrays of non-Watson–Crick basepairs that in the secondary structure representation form hairpin or internal loops, multi-stem junctions, and even pseudoknots. Although RNA motifs occur recurrently and contribute in a modular fashion to RNA architecture, it is usually not apparent which bases interact and whether it is by edge-to-edge H-bonding or solely by stacking interactions. Using a modular sequence-analysis approach, recurrent motifs related to the sarcin–ricin loop of 23S RNA and to loop E from 5S RNA were predicted in universally conserved regions of the large ribosomal RNAs (16S- and 23S-like) before the publication of high-resolution, atomic-level structures of representative examples of 16S and 23S rRNA molecules in their native contexts. This provides the opportunity to evaluate the predictive power of motif-level sequence analysis, with the goal of automating the process for predicting RNA motifs in genomic sequences. The process of inferring structure from sequence by constructing accurate alignments is a circular one. The crucial link that allows a productive iteration of motif modeling and realignment is the comparison of the sequence variations for each putative pair with the corresponding isostericity matrix to determine which basepairs are consistent both with the sequence and the geometrical data.

Observation of H-bond mediated 3hJH2H3 coupling constants across Watson-Crick AU base pairs in RNA.

3hJH2H3 trans-hydrogen bond scalar coupling constants have been observed for the first time in Watson-Crick AU base pairs in uniformly 15N-labeled RNA oligonucleotides using a new 2hJNN-HNN-E. COSY experiment. The experiment utilizes adenosine H2 (AH2) for original polarization and detection, while employing 2hJNN couplings for coherence transfer across the hydrogen bonds (H-bonds). The H3 protons of uracil bases are unperturbed throughout the experiment so that these protons appear as passive spins in E. COSY patterns. 3hJH2H3 coupling constants can therefore be accurately measured in the acquisition dimension from the displacement of the E. COSY multiplet components, which are separated by the relatively large 1JH3N3 coupling constants in the indirect dimension of the two-dimensional experiment. The 3hJH2H3 scalar coupling constants determined for AU base pairs in the two RNA hairpins examined here have been found to be positive and range in magnitude up to 1.8 Hz. Using a molecular fragment representation of an AU base pair, density functional theory/finite field perturbation theory (DFT/FPT) methods have been applied to attempt to predict the relative contributions of H-bond length and angular geometry to the magnitude of 3hJH2H3 coupling constants. Although the DFT/FPT calculations did not reproduce the full range of magnitude observed experimentally for the 3hJH2H3 coupling constants, the calculations do predict the correct sign and general trends in variation in size of these coupling constants. The calculations suggest that the magnitude of the coupling constants depends largely on H-bond length, but can also vary with differences in base pair geometry. The dependency of the 3hJH2H3 coupling constant on H-bond strength and geometry makes it a new probe for defining base pairs in NMR studies of nucleic acids.

Adenine recognition: a motif present in ATP-, CoA-, NAD-, NADP-, and FAD-dependent proteins.

Adenosine triphosphate (ATP) plays an essential role in energy transfer within the cell. In the form of NAD, adenine participates in multiple redox reactions. Phosphorylation and ATP-hydrolysis reactions have key roles in signal transduction and regulation of many proteins, especially enzymes. In each cell, proteins with many different functions use adenine and its derivatives as ligands; adenine, of course, is present in DNA and RNA. We show that an adenine binding motif, which differs according to the backbone chain direction of a loop that binds adenine (and in one variant by the participation of an aspartate side-chain), is common to many proteins; it was found from an analysis of all adenylate-containing protein structures from the Protein Data Bank. Indeed, 224 protein-ligand complexes (86 different proteins) from a total of 645 protein structure files bind ATP, CoA, NAD, NADP, FAD, or other adenine-containing ligands, and use the same structural elements to recognize adenine, regardless of whether the ligand is a coenzyme, cofactor, substrate, or an allosteric effector. The common adenine-binding motif shown in this study is simple to construct. It uses only (1) backbone polar interactions that are not dependent on the protein sequence or particular properties of amino acid side-chains, and (2) nonspecific hydrophobic interactions. This is probably why so many different proteins with different functions use this motif to bind an adenylate-containing ligand. The adenylate-binding motif reported is present in "ancient proteins" common to all living organisms, suggesting that adenine-containing ligands and the common motif for binding them were exploited very early in evolution. The geometry of adenine binding by this motif mimics almost exactly the geometry of adenine base-pairing seen in DNA and RNA.

Intercalation of the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence: DNA sequence effects.

The structure of the bay region (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X(7) of 5'-d(CGGACAXGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined by NMR. This was the bay region benz[a]anthracene RSRS (61,3) adduct. The BA moiety intercalated above the 5'-face of the modified base pair. NOE connectivities between imino protons were disrupted at T16 and T17. Large chemical shifts at the lesion site were consistent with ring current shielding arising from the BA moiety. A large chemical shift dispersion was observed for the BA aromatic protons. An increased rise of 8.17 A was observed between base pairs A6 x T17 and X7 x T(16). The PAH moiety stacked with the purine ring of A6, the 5'-neighbor nucleotide. This resulted in buckling of the 5'-neighbor A6 x T17 base pair, evidenced by exchange broadening for the T17 imino resonance. It also interrupted sequential NOE connectivities between nucleotides C5 and A6. The A6 deoxyribose ring showed an increased percentage of the C3'-endo conformation. This differed from the bay region BA RSRS (61,2) adduct, in which the lesion was located at position X6 [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981], but was similar to the benzo[a]pyrene BP SRSR (61,3) adduct [Zegar I. S., Chary, P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd, R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Biochemistry 37, 16516-16528]. The altered sugar pseudorotation at A6 appears to be common to both bay region BA RSRS (61,3) and BP SRSR (61,3) adducts. It could not be discerned if the C3'-endo conformation at A6 in the BA RSRS (61,3) adduct altered base pairing geometry at X7 x T16, as compared to the C2'-endo conformation. The structural studies suggest that the mutational spectrum of this adduct may be more complex than that of the BA RSRS (61,2) adduct.

Recognition of Cognate Transfer RNA by the 30 S Ribosomal Subunit

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.

Structure-Function Relationship of Three Triple-Helical Nucleic Acids

The nucleic acid triplexes poly d(T)·poly d(A)·poly d(T), poly (U)·poly (A)·poly (U), and poly (I)·poly (A)·poly (I) display a sort of continuity between each other. However, their morphologies present their own individuality which, considering those of their parent duplexes, are quite unexpected. This comparison helps to understand triplex structure-function relationship. While helical parameters are functions of the sugar pucker, low values of WC and Hoogsteen base-pair propellers is commonplace for triplexes and the Hoogsteen base-pair geometry monitors the effects of the interstrand phosphates charge-charge repulsion. Synopsis The nucleic acid triplexes poly d(T)·poly d(A)·poly d(T), poly(U)·poly(A)·poly(U), and poly (I)·poly (A)·poly (I) present distinct morphologies. Considering those of their parent duplexes, they are also quite unexpected.

A nicked duplex decamer DNA with a PEG(6) tether.

A dumbbell double-stranded DNA decamer tethered with a hexaethylene glycol linker moiety (DDSDPEG), with a nick in the centre of one strand, has been synthesised. The standard NMR methods, E.COSY, TOCSY, NOESY and HMQC, were used to measure (1)H, (31)P and T:(1) spectral parameters. Molecular modelling using rMD-simulated annealing was used to compute the structure. Scalar couplings and dipolar contacts show that the molecule adopts a right-handed B-DNA helix in 38 mM phosphate buffer at pH 7. Its high melting temperature confirms the good base stacking and stability of the duplex. This is partly attributed to the presence of the PEG(6) linker at both ends of the duplex that restricts the dynamics of the stem pentamers and thus stabilises the oligonucleotide. The inspection of the global parameters shows that the linker does not distort the B-DNA geometry. The computed structure suggests that the presence of the nick is not disturbing the overall tertiary structure, base pair geometry or duplex base pairing to a substantial extent. The nick has, however, a noticeable impact on the local geometry at the nick site, indicated clearly by NMR analysis and reflected in the conformational parameters of the computed structure. The (1)H spectra also show much sharper resonances in the presence of K(+) indicating that conformational heterogeneity of DDSDPEG is reduced in the presence of potassium as compared to sodium or caesium ions. At the same time the (1)H resonances have longer T:(1) times. This parameter is suggested as a sensitive gauge of stabilisation.

Significance of Nucleobase Shape Complementarity and Hydrogen Bonding in the Formation and Stability of the Closed Polymerase−DNA Complex

DNA polymerases insert a dNTP by a multistep mechanism that involves a conformational rearrangement from an open to a closed ternary complex, a process that positions the incoming dNTP in the proper orientation for phosphodiester bond formation. In this work, the importance and relative contribution of hydrogen-bonding interactions and the geometric shape of the base pair that forms during this process were studied using Escherichia coli DNA polymerase I (Klenow fragment, 3'-exonuclease deficient) and natural dNTPs or non-hydrogen-bonding dNTP analogues. Both the geometric fit of the incoming nucleotide and its ability to form Watson-Crick hydrogen bonds with the template were found to contribute to the stability of the closed ternary complex. Although the formation of a closed complex in the presence of a non-hydrogen-bonding nucleotide analogue could be detected by limited proteolysis analysis, a comparison of the stabilities of the ternary complexes indicated that hydrogen-bonding interactions between the incoming dNTP and the template increase the stability of the complex by 6-20-fold. Any deviation from the Watson-Crick base pair geometry was shown to have a destabilizing effect on the closed complex. This degree of destabilization varied from 3- to 730-fold and was found to be correlated with the size of the mismatched base pair. Finally, a stable closed complex is not formed in the presence of a ddNTP or rNTP. These results are discussed in relation to the steric exclusion model for the nucleotide insertion.

An ab Initio Study of Hydrogen Bonding Effects on the 15N and 1H Chemical Shielding Tensors in the Watson−Crick Base Pairs

Density functional theory is applied to explore changes upon hydrogen bonding in the 15N and 1H nuclear magnetic resonance (NMR) chemical shielding tensors of the imino group in the Watson−Crick nucleic acid base pairs. The dependence of results on the quantum chemical method used, on the basis set superposition error, and on the effect of the relaxation of the base pair geometry with changing hydrogen bond length is addressed. The systematic variation with changing separation between bases in calculated shielding data and resulting auto- and cross-corelation chemical shift anisotropy parameters is documented. Possible implications for NMR studies of the dynamics of 15N−1H bond vectors and for transverse relaxation-optimized spectroscopy experiments on imino protons are discussed.

Water-Mediated Base Pairs in RNA: A Quantum-Chemical Study

We have studied the geometric and energetic properties of the six water-mediated base pairs WUC, WGA, WUA, WUU, WUG, WGG, (W: water) and of the related water-free complexes by quantum-chemical calculations including electron correlation at the MP2/6-31G(d,p)//HF/6-31G(d,p) and B3LYP/6-31G(d,p) levels. In the water-mediated WUC, WGA ,and WUU base pairs, water is both donor and acceptor of H-bonds, and for these complexes the calculated hydrogen bonding patterns are close to the experimental ones within the RNA helix and allow cooperative effects within the H-bond network. The geometries of these base pairs are obviously almost not affected by the nucleic acid and solvent environment. Therefore, they can be considered as structurally autonomous building blocks of RNA. In the optimized structure of the WUA base pair water still links the two bases, yet the H-bond pattern deviates somewhat from the one in the RNA crystal structure and it is not cooperative. Therefore, we classify the WUA base pair as an intermediate case. In WUG and WGG pairs, water is twice acceptor or twice donor. These complexes are not structurally autonomous, but apparently have to be stabilized by additional interactions with the surrounding nucleic acid and solvent. For the UG pair a water-mediated C5−H5(U)···O6(G) contact involving an alternative water molecule (371) in the major groove is important and leads to a base pair geometry resembling the experimental structure (W371UG). In the GG pair, short contacts to a backbone C5‘-H5‘ donor group and to a further water molecule are likely to stabilize the experimental base pair geometry. Similar to exocyclic amino groups, water involved in base pairing induces nonplanar equilibrium geometries, yet except for one of the examples (WGA) the energy difference to the corresponding planar conformation is very small. Cooperative effects have been found to contribute between 9 and 13% at the MP2/HF level and between 8 and 20% at the B3LYP level to the total interaction energy of all water-mediated pairs except for WUA. WUC is the only example for which the absolute value of the total interaction energy per H-bond is significantly increasing in passing from the direct to the water-mediated pair.

Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases.

Recent studies have identified amino acid side chains forming several hydrogen bonds in the DNA minor groove as potentially important in polymerase replication of DNA. Few studies have probed these interactions on the DNA itself. Using non-hydrogen-bonding nucleoside isosteres, we have now studied effects in both primer and template strands with several polymerases to investigate the general importance of these interactions. All six polymerases show differences in the H-bonding effects in the minor groove. Two broad classes of activity are seen, with a first group of DNA polymerases (KF(-), Taq, and HIV-RT) that efficiently extends nonpolar base pairs containing nucleoside Q (9-methyl-1H-imidazo[4,5-b]pyridine) but not the analogue Z (4-methylbenzimidazole), implicating a specific minor groove interaction at the first extension site. A second group of polymerases (Pol alpha, Pol beta, and T7(-)) fails to extend all non-H-bonding base pairs, indicating that these enzymes may need minor groove hydrogen bonds at both minor groove sites or that they are especially sensitive to noncanonical DNA structure or stability. All DNA polymerases examined use energetically important minor groove interactions to probe newly synthesized base pairs before extending them. The positions of these interactions vary among the enzymes, and only a subset of the interactions identified structurally appears to be functionally important. In addition, polymerases appear to be differently sensitive to small changes in base pair geometry.