DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.     

Crystal structures of a ddATP-, ddTTP-, ddCTP, and ddGTP- trapped ternary complex of Klentaq1: insights into nucleotide incorporation and selectivity

The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP). In this report, we present the details of the 2.3-A resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP), or a dideoxyadenosine 5'-triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson-Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen-bonding interactions may provide a means of detecting misincorporation at this position.     

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.     

Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta

Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase eta (Poleta) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Poleta are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Poleta; in this regard, Poleta differs strikingly from classical high-fidelity DNA polymerases.     

Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli

The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.     

Varying DNA base-pair size in subangstrom increments: evidence for a loose, not large, active site in low-fidelity Dpo4 polymerase

We describe the first systematic test of steric effects in the active site of a Y-family DNA polymerase, Dpo4. It has been hypothesized that low-fidelity repair polymerases in this family more readily accept damaged or mismatched base pairs because of a sterically more open active site, which might place lower geometric constraints on the incipient pair. We have tested the origin of low fidelity by use of five nonpolar thymidine analogues that vary in size by a total of 1.0 A over the series. The efficiency and fidelity of base-pair synthesis was measured by steady-state kinetics for single-nucleotide insertions. Analogues were examined both as incoming deoxynucleoside triphosphate (dNTP) derivatives and as template bases. The results showed that Dpo4 preferred to pair the thymidine shape mimics with adenine and, surprisingly, the preferred size was at the center of the range, the same optimum size as recently found for the high-fidelity Klenow fragment (Kf) of Escherichia coli DNA Pol I. However, the size preference with Dpo4 was quite small, varying by a factor of only 30-35 from most to least efficient thymidine analogue. This is in marked contrast to Kf, which showed a rigid size preference, varying by 1100-fold from best to worst. The fidelity for the non-hydrogen-bonding analogues in pairing with A over T, C, or G was much lower in Dpo4 than in the previous high-fidelity enzyme. The data establish that, unlike Kf, Dpo4 has very low steric selectivity and that steric effects alone cannot explain the fidelity (albeit low) that Dpo4 has for a correct base pair; the findings suggest that hydrogen bonds may be important in determining the fidelity of this enzyme. The results suggest that the low steric selectivity of this enzyme is the result of a conformationally flexible or loose active site that adapts with small energetic cost to different base-pair sizes (as measured by the glycosidic C1'-C1' distance), rather than a spatially large active site.     

Human DNA primase uses Watson-Crick hydrogen bonds to distinguish between correct and incorrect nucleoside triphosphates

Human DNA primase synthesizes short RNA primers that DNA polymerase alpha further elongates. Primase readily misincorporates the natural NTPs and will generate a wide variety of mismatches. In contrast, primase exhibited a remarkable resistance to polymerizing NTPs containing unnatural bases. This included bases whose shape was almost identical to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base [e.g., 5- and 6-(trifluoromethyl)benzimidazole], bases much more hydrophobic than a natural base [e.g., 4- and 7-(trifluoromethyl)benzimidazole], bases of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-D-guanine), and bases capable of forming only one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). Primase only polymerized NTP analogues containing bases capable of forming hydrogen bonds between the equivalent of both N-1 and the exocyclic group at C-6 of a purine NTP (2-fluoroadenine, 2-chloroadenine, 3-deazaadenine, and hypoxanthine) and N-3 and the exocyclic group at C-4 of a pyrimidine. These data indicate that human primase requires the formation of Watson-Crick hydrogen bonds in order to polymerize a NTP, a situation very different than what is observed with some DNA polymerases. The implications of these results with respect to current theories of how polymerases discriminate between right and wrong (d)NTPs are discussed.     

Structural insights into DNA polymerase beta deterrents for misincorporation support an induced-fit mechanism for fidelity

DNA polymerases generally select the correct nucleotide from a pool of structurally similar molecules to preserve Watson-Crick base-pairing rules. We report the structure of DNA polymerase beta with DNA mismatches situated in the polymerase active site. This was achieved by using nicked product DNA that traps the mispair (template-primer, A-C or T-C) in the nascent base pair binding pocket. The structure of each mispair complex indicates that the bases do not form hydrogen bonds with one another, but form a staggered arrangement where the bases of the mispair partially overlap. This prevents closure/opening of the N subdomain that is believed to be required for catalytic cycling. The partially open conformation of the N subdomain results in distinct hydrogen bonding networks that are unique for each mispair. These structures define diverse molecular aspects of misinsertion that are consistent with the induced-fit model for substrate specificity.     

Structures of Oligonucleotides Containing 5-(methoxymethyl)-2′-deoxyuridine Determined by NMR Spectroscopy

Abstract

Base pairing of 5-(methoxymethyl)-2′-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU. Abase pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH₂ and CH₂?O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU. G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.     

Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa

The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Poleta, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polkappa, which differs from Poleta in having a higher fidelity and which, unlike Poleta, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Poleta. We find that an isosteric thymidine analog is replicated with low efficiency by Polkappa, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Poleta and Polkappa rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.     

Structural diversity and isomorphism of hydrogen-bonded base interactions in nucleic acids

The wide structural diversity of RNA results in part from the diversity of non-Watson-Crick interactions between bases. To examine the repertoire of possible hydrogen bond interactions among bases, we computed databases of base-pairs and base-triples by systematically matching all possible hydrogen-bond donors and acceptors between bases and evaluating the geometries of each planar configuration. For base-pairs, we find 53 arrangements having at least two hydrogen bonds, including 23 pairs with protonated bases that have not previously been modeled. A comparison with experimentally observed base-pairs reveals an unexpected G:U pair recently observed in the ribosome. For base-triples, we find 840 arrangements in which the three bases are constrained by a total of at least three hydrogen bonds. Base-triples in particular exhibit a wide range of structural diversity, suggesting how compact or elongated nucleic acid structures may be constructed using different hydrogen-bonding patterns. Base-pair and base-triple conformations were systematically compared to identify structurally isomorphic combinations, and the experimentally observed arrangements within double and triple helices are among the most isomorphic. Unexpectedly, however, other combinations in the database are even more isomorphic, including several in which all-purine arrangements overlap with all-pyrimidine arrangements. These studies highlight some of the combinatoric and geometric versatility of base interactions and help provide a framework for analyzing and modeling isomorphic interactions and potentially for designing novel nucleic acid structures.     

Conformation of guanine-8-oxoadenine base pairs in the crystal structure of d(CGCGAATT(O8A)GCG).

The structure of the synthetic deoxydodecamer d(CGCGAATT(O8A)GCG)2 (O8A = 8-oxoadenine) has been determined by single-crystal X-ray diffraction techniques. The oligonucleotide crystallizes in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 25.48 A, b = 41.84 A, and c = 64.91 A. The refinement has converged with an R-factor of 0.151 for 1119 reflections in the resolution range 8.0-2.25 A. Sixty-seven solvent molecules were located during the course of the refinement. The B-DNA helix consists of ten Watson-Crick base pairs and two guanine-8-oxoadenine (G.O8A) base pairs. In order to achieve hydrogen-bonding complementarity between the two bases, an unusual G(anti).O8A-(syn) wobble conformation is adopted. It is proposed that the G.O8A mispairs are held together by a network of four interbase hydrogen bonds which are the result of the formation of two reverse three-center hydrogen-bonding systems. These involve one carbonyl oxygen lone pair interacting with two hydrogen atoms. In a departure from previous observations of the characteristics of purine-purine anti-syn base pairs, lambda 1 and lambda 2, the angles between the glycosidic bonds and the C1'-C1' vector, are symmetric. A reassessment of the other purine-purine mispairs suggests that similar three-center hydrogen bonds may occur and make a contribution to stabilizing other base pairings.     

Effects of the minor groove pyrimidine nucleobase functional groups on the stability of duplex DNA: the impact of uncompensated minor groove amino groups

DNA sequences containing four types of analog nucleosides are described. All four are pyridine derivatives constructed as C-nucleosides so that they mimic the pyrimidine derivatives 2'-deoxyuridine, thymidine or 2'-deoxycytidine, but in all cases the analogs lack the corresponding O2-carbonyls that in duplex DNA are located in the minor groove. In place of the O2-carbonyl is a hydrogen atom, a polar fluorine atom, or a nonpolar methyl group. The described C-nucleosides have native-like bidentate Watson-Crick hydrogen-bonding faces and can form essentially normal W-C base pairs of varying stability with A or G. In each modified base pair, two inter-residue hydrogen bonds should be present. In spite of a common number of interstrand hydrogen bonds, the thermodynamic stabilities of the prepared duplexes, each containing two analog base pairs, vary dramatically. Most notably, base pairs containing uncompensated purine amino groups (those lacking a hydrogen-bonding partner) in the minor groove exhibit the most dramatic reductions in thermodynamic stability. Removal of such uncompensated amino groups results in increased duplex stability. Base pairs containing fluorine in the minor groove positioned adjacent to an amino group seem to enhance duplex stability marginally (relative to --H or --CH(3)), but there is little evidence to suggest that fluorine is an effective hydrogen-bonding partner in these systems. The presence of minor groove methyl groups results in the least stable duplexes in each series of sequences.     

Solution structure of an oncogenic DNA duplex containing a G.A mismatch

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.     

Influence of local sequence context on damaged base conformation in human DNA polymerase ι: molecular dynamics studies of nucleotide incorporation opposite a benzo[a]pyrene-derived adenine lesion

Human DNA polymerase ι is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions in both near error-free and error-prone bypass. With undamaged templating purines polymerase ι normally favors Hoogsteen base pairing. Polymerase ι can incorporate nucleotides opposite a benzo[a]pyrene-derived adenine lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. We performed molecular modeling and molecular dynamics simulations to elucidate the structural basis for lesion bypass. Our results suggest that hydrogen bonds between the benzo[a]pyrenyl moiety and nearby bases limit the movement of the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5′-CAGA*TT-3′ sequence. This facilitates correct incorporation of dT via a Watson−Crick pair. In a 5′-TTTA*GA-3′ sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA*, there is also an opportunity for increased misincorporation of dGTP. These results expand our understanding of the versatility and flexibility of polymerase ι and its lesion bypass functions in humans.     

A stable antiparallel cytosine-thymine base pair occurring only at the end of a duplex.

Ultraviolet hyperchromicity experiments indicate that in DNA duplex formation, a C-T mismatch is destabilizing in the center of a duplex, but behaves as a stable base pair at the terminus of a duplex. The C-T base pair is thought to contain two hydrogen bonds, but has thermodynamic parameters (delta Ho and delta Go of dissociation) that are similar to a G-C base pair. AMBER molecular mechanics calculations were performed to study the possible structural properties of DNA duplexes with central and terminal C-T combinations. These calculations also indicate that a central C-T pair destabilizes a duplex, while terminal C-T forms a stable base pair. Hydrogen bonding between cytosine and thymine occurs only in the energy-minimized structures when the helix diameter decreases and the propeller twist angle between the bases increases. These changes are found to occur only at the end of a duplex in the calculations, which may explain the experimental results.     

A conformational rationale for the replication of nucleic acids under prebiotic conditions

The Watson-Crick model at once gave an explanation for the mechanism of replication of DNA. But the hydrogen-bonding forces between the bases alone are not enough for the specificity of base-pairing mechanisms, since any pair of bases can be positioned to have at least two hydrogen bonds. In the present-day biological organisms, sophisticated enzymatic machinery is supposed to constrain the ribose-phosphate backbone to have regular structure, aiding the self-templating duplication. For the prebiotic stage, whence sophisticated enzymes would not have been evolved, we propose a novel double helical conformation of DNA wherein the two sugar-phosphate backbones are pulled towards each other by (CH  O) hydrogen bonds conferring stereospecificity for the formation of (A : T)- and (G : C)-pairs, in the self-templating chains of DNA. Our model-building efforts and computer calculations endorse the stereochemical feasibility of the conformation. The pairing of homologous sequences of two double helices of DNA is explained by direct hydrogen-bonding interactions in our model and it is thus relevant to the present-day biological functions also, at least in some stages of the cell-cycle.     

Herpes simplex virus 1 primase employs watson-crick hydrogen bonding to identify cognate nucleoside triphosphates

We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-d-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethylbenzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed.     

Theoretical studies on protein-nucleic acid interactions. II. Hydrogen bonding of amino acid side chains with bases and base pairs of nucleic acids

With a view to understanding the role of hydrogen bonds in the recognition of nucleic acids by proteins, hydrogen bonding between the bases and base pairs of nucleic acids and the amino acids (Asn, Gln, Asp and Glu, and charged residues Arg⁺, Glu^?, and Asp^?) has been studied by a second-order perturbation theory. Binding energies have been calculated for all possible configurations involving a pair of hydrogen bonds between the base (or base pair) and the amino acid residue. Our results show that the hydrogen bonding in these cases has a large contribution from electrostatic interaction. In general, the charged amino acids, compared to the uncharged ones, form more stable complexes with bases or base pairs. The hydrogen-bond energies are an order of magnitude smaller than the Coulombic interaction energies between basic amino acids (Lys⁺, Arg⁺, and His⁺) and the phosphate groups of nucleic acids. The stabilities of the complexes of amino acids Asn, Gln, Asp, and Glu with bases are in the order: G–X > C–X > A–X U–X or T–X, and G · C–X > A · T(U)–X, where X is one of these amino acid residues. It has been shown that Glu^? and Asp^? can recognize guanine in single-stranded nucleic acids; Arg⁺ can recognize G · C base pairs from A · T base pairs in double-stranded structures.