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.     

Out-of-shape DNA minor groove binders: induced fit interactions of heterocyclic dications with the DNA minor groove

DB921 and DB911 are benzimidazole-biphenyl isomers with terminal charged amidines. DB911 has a central meta-substituted phenyl that gives it a shape similar to those of known minor groove binding compounds. DB921 has a central para-substituted phenyl with a linear conformation that lacks the appropriate radius of curvature to match the groove shape. It is thus expected that DB911, but not DB921, should be an effective minor groove binder, but we find that DB921 not only binds in the groove but also has an unusually high binding constant in SPR experiments (2.9 x 10(8) M(-)(1), vs 2.1 x 10(7) M(-)(1) for DB911). ITC thermodynamic analysis with an AATT sequence shows that the stronger binding of DB921 is due to a more favorable binding enthalpy relative to that of DB911. CD results support minor groove binding for both compounds but do not provide an explanation for the binding of DB921. X-ray crystallographic analysis of DB921 bound to AATT shows that an induced fit structural change in DB921 reduces the twist of the biphenyl to complement the groove, and places the functional groups in position to interact with bases at the floor of the groove. The phenylamidine of DB921 forms indirect contacts with the bases through a bound water. The DB921-water pair forms a curved binding module that matches the shape of the minor groove and provides a number of strong interactions that are not possible with DB911. This result suggests that traditional views of compound curvature required for minor groove complex formation should be reevaluated.     

Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA. Evidence for both major and minor groove contacts

Beta-D-Glucosyl-hydroxymethyluracil, also called base J, is an unusually modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously identified a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia, and we have shown that it is a structure-specific binding protein. Here we examine the molecular interactions that contribute to recognition of the glycosylated base in synthetic DNA substrates using modification interference, modification protection, DNA footprinting, and photocross-linking techniques. We find that the two primary requirements for J-DNA recognition include contacts at base J and a base immediately 5' of J (J-1). Methylation interference analysis indicates that the requirement of the base at position J-1 is due to a major groove contact independent of the sequence. DNA footprinting of the JBP.J-DNA complex with 1,10-phenanthroline-copper demonstrates that JBP contacts the minor groove at base J. Substitution of the thymine moiety of J with cytosine reduces the affinity for JBP approximately 15-fold. These data indicate that the sole sequence dependence for JBP binding may lie in the thymine moiety of base J and that recognition requires only two specific base contacts, base J and J-1, within both the major and minor groove of the J-DNA duplex.     

Thallium-DNA complexes in aqueous solution. Major or minor groove binding

Thallium (Tl) binds to the major and minor grooves of B-DNA in the solid state (Howerton et al., Biochemistry 40, 10023-10031, 2001). The aim of this study was to examine the binding of Tl(I) cation with calf-thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (12.5 mM) and various concentrations of metal ions (0.5 to 20 mM). UV-visible and FTIR spectroscopic methods were used to determine the cation binding site, the binding constant and DNA structural variations in aqueous solution. Direct Tl bindings to guanine and thymine were evident by major spectral changes of DNA bases with overall binding constant of K = 1.40 x 10(4) M(-1) and little perturbations of the backbone phosphate group. Both major and minor groove bindings were observed with no alteration of the B-DNA conformation. At low metal concentration (0.5 mM), the number of cations bound were 10 per 1000 nucleotides, while at higher cation concentration (10 mM), this increased to 30 cations per 1000 nucleotides.     

DNA minor groove interactions and the biological activity of 2,5-bis

2,5-Bis-[4-(N-cyclobutyl-amidino)phenyl] furan and 2,5-bis-[4-(N-cyclohexyl-amidino)phenyl] furan have activity against Pneumocystis carinii and also show cytotoxicity against several tumour cell lines. These activities are correlated with DNA-binding abilities; the crystal structures of complexes with the DNA sequence d(CGCGAATTCGCG) is reported here. Interactions with, and effects on, the DNA minor groove, are found to be factors in the biological properties of these compounds.     

Probing minor groove hydrogen bonding interactions between RB69 DNA polymerase and DNA

Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2'-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10(2)-10(3)-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10(2)-10(3)-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n - 2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.     

Molecular modelling of ligand-DNA minor groove binding: role of ligand-water interactions

A procedure was developed for quantitative estimation of the ligand affinity for the DNA minor groove with allowance for ligand hydration, whereby the binding energy was calculated as the difference in the energies of ligand-DNA and ligand-water interactions. Adequacy of the procedure was demonstrated with the structural motifs (pyrrolecarboxamide, benzimidazole, furancarboxamide, and phthalimide) of well-known ligands for the case of a d(GCA10CG).d(CGT10GC) duplex. On the strength of the results obtained, an indole-based motif was proposed as the basis for a highly affined minor groove binder.     

Direct comparison of A- and T-strand minor groove interactions in DNA curvature at A tracts

To investigate the relative contributions of minor-groove electrostatic interactions in the mechanism of A-tract DNA curvature, we carried out experiments with modified DNA bases in both strands of the tract. We employed 3-deazaadenine nucleoside (D), which lacks the adenine N3 nitrogen in the minor groove and thus cannot act as an electron donor, as well as difluorotoluene (F), a nonpolar thymine mimic. The effects of these analogues in A-tract curvature were quantified using ligation ladder gel mobility methods developed by Crothers and by Maher. Through single substitutions of D in A(5) tracts, we found that this analogue results in decreased curvature only when situated toward the 3' end of the tract. This is distinct from the behavior in the T-rich strand where F substitution causes the greatest reductions in curvature toward the 5' end. To test for cooperative pairwise effects, we also studied 10 different D + F double substitutions and found evidence supporting a number of localized cooperative electrostatic interactions but not between the two most sensitive sites in the opposite strands. These results suggest that there are two discrete locations in the A-tract minor groove where electrostatic interactions are important in causing curvature: one near the 5' end of the T-rich strand, and one near the 3' end of the A-rich strand. The results are consistent with an important role of localized cations in the minor groove. Possible effects of groove solvation and stacking at the A-tract junction are also discussed.     

Fluorescent d(CGCGAATTCGCG): characterization of major groove polarity and study of minor groove interactions through a major groove semantophore conjugate.

The major and minor groove in duplex DNA are sites of specific molecular recognition by DNA-binding agents such as proteins, drugs and metal complexes and have functional significance. In view of this, understanding of the inherent differences in their environment and the allosteric information transfer between them induced by DNA-binding agents assumes importance. Site-specific incorporation of 5-aminodansyl-dU, (U*) in oligonucleotides d(CGCGAAU*TCGCG) and d(CGCGAATU*CGCG) leads to fluorogenic nucleic acids, in which the reporter group resides in the major groove. The fluorescent observables from such a probe are used to estimate the dielectric constant of the major groove to be approximately 55D, in comparison to the reported non polar environment of the minor groove (approximately 20D) in poly d[AT]-poly d[AT]. An exclusive minor groove event such as DNA-netropsin association can be quantitatively monitored by fluorescence of the dansyl moiety located in the major groove. This suggests existence of an information network among the two grooves. The fluorescent DNA probes as reported here may have potential applications in the study of structural polymorphisms in DNA, DNA-ligand interactions and triple helix structure.     

DNA A-tracts bending: polarization effects on electrostatic interactions across their minor groove

Bending by the DNA A-tracts constitutes a contentious issue, suggesting deficiencies in the physics employed so far. Here, we inquire as to the importance in this bending of many-body polarization effects on the electrostatic interactions across their narrow minor groove. We have done this on the basis of the findings of Jarque and Buckingham who developed a procedure based on a Monte Carlo simulation for two charges of the same sign embedded in a polarizable medium. Remarkably, the present analysis reveals that for compact DNA conformations, which result from dynamic effects, an overall attractive interaction operates between the phosphate charges; this interaction is especially strong for the narrow minor groove of the A-tracts, suggesting a tendency for DNA to bend toward this groove. This tendency is in agreement with the conclusions of electrophoretic and NMR solution studies. The present analysis is also consistent with the experimental observations that the minor groove is much more easily compressible than the major groove and the bending propensity of the A-tracts is greatly reduced at “premelting” temperatures. By contrast, the dielectric screening model predicts a repulsion between the phosphate charges and is not consistent with the aforementioned bending tendency or experimental observations.     

FTIR study of specific binding interactions between DNA minor groove binding ligands and polynucleotides.

The use of FTIR spectroscopy is made to study the interactions between polynucleotides and two series of minor groove binding compounds. The latter were developed and described previously as part of an ongoing program of rational design of modified ligands based on naturally occurring pyrrole amidine antibiotic netropsin, and varying the structure of bisbenzimidazole chromosomal stain Hoechst 33258. Characteristic IR absorptions due to the vibrations of thymidine and cytosine keto groups in polynucleotides containing AT and GC base pairs respectively are used to monitor their interaction with the added ligands. Although the two thiazole based lexitropsins based on netropsin structure differ in the relative orientation of nitrogen and sulfur atoms with respect to the concave edge of the molecules, they interact exclusively with the thymidine C2 = O carbonyl groups in the minor groove of the alternating AT polymer as evidenced by specific changes in the IR spectra. In the second series of compounds based on Hoechst 33258, the structure obtained by replacing the two benzimidazoles in the parent compound by a combination of pyridoimidazole and benzoxazole, exhibits changes in the carbonyl frequency region of poly dG.poly dC which is attributed to the ligand interaction at the minor groove of GC base pairs. In contrast, Hoechst 33258 itself interacts only with poly dA.poly dT. Weak or no interaction exists between the ligands and any of the polynucleotides at the levels of the phosphate groups or the deoxyribose units.     

Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove.

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.     

The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.

I-TevI, a double-strand DNA endonuclease encoded by the mobile td intron of phage T4, has specificity for the intronless td allele. Genetic and physical studies indicate that the enzyme makes extensive contacts with its DNA substrate over at least three helical turns and around the circumference of the helix. Remarkably, no single nucleotide within a 48 bp region encompassing this interaction domain is essential for cleavage. Although two subdomains (DI and DII) contain preferred sequences, a third domain (DIII), a primary region of contact with the enzyme, displays much lower sequence preference. While DII and DIII suffice for recognition and binding of I-TevI, all three domains are important for formation of a cleavage-competent complex. Mutational, footprinting and interference studies indicate predominant interactions of I-TevI across the minor groove and phosphate backbone of the DNA. Contacts appear not to be at the single nucleotide level; rather, redundant interactions and/or structural recognition are implied. These unusual properties provide a basis for understanding how I-TevI recognizes T-even phage DNA, which is heavily modified in the major groove. These recognition characteristics may increase the range of natural substrates available to the endonuclease, thereby extending the invasive potential of the mobile intron.     

Sequence and length dependent thermodynamic differences in heterocyclic diamidine interactions at AT base pairs in the DNA minor groove

With the goal of developing a better understanding of the antiparasitic biological action of DB75, we have evaluated its interaction with duplex alternating and nonalternating sequence AT polymers and oligomers. These DNAs provide an important pair of sequences in a detailed thermodynamic analysis of variations in interaction of DB75 with AT sites. The results for DB75 binding to the alternating and nonalternating AT sequences are quite different at the fundamental thermodynamic level. Although the Gibbs energies are similar, the enthalpies for DB75 binding with poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) are +3.1 and -4.5 kcal/mol, respectively, while the binding entropies are 41.7 and 15.2 cal/mol.K, respectively. The underlying thermodynamics of binding to AT sites in the minor groove plays a key role in the recognition process. It was also observed that DB75 binding with poly(dA).poly(dT) can induce T.A.T triplet formation and the compound binds strongly to the dT.dA.dT triplex.     

Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications

Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.