Insights into the A-C Mismatch Conformational Ensemble in Duplex DNA and its Role in Genetic Processes through a Structure-based Review

Abstract

Knowing the conformational ensembles formed by mismatches is crucial for understanding how they are generated and repaired and how they contribute to genomic instability. Here, we review structural and energetic studies of the A-C mismatch in duplex DNA and use the information to identify critical conformational states in its ensemble and their significance in genetic processes. In the 1970s, Topal and Fresco proposed the A-C wobble stabilized by two hydrogen bonds, one requiring protonation of adenine-N1. Subsequent NMR and X-ray crystallography studies showed that the protonated A-C wobble was in dynamic equilibrium with a neutral inverted wobble. The mismatch was shown to destabilize duplex DNA in a sequence- and pH-dependent manner by 2.4-3.8 kcal/mol and to have an apparent pKa ranging between 7.2 and 7.7. The A-C mismatch conformational repertoire expanded as structures were determined for damaged and protein-bound DNA. These structures included Watson-Crick-like conformations forming through tautomerization of the bases that drive replication errors, the reverse wobble forming through rotation of the entire nucleotide proposed to increase the fidelity of DNA replication, and the Hoogsteen base-pair forming through the flipping of the adenine base which explained the unusual specificity of DNA polymerases that bypass DNA damage. Thus, the A-C mismatch ensemble encompasses various conformational states that can be selectively stabilized in response to environmental changes such as pH shifts, intermolecular interactions, and chemical modifications, and these adaptations facilitate critical biological processes. This review also highlights the utility of existing 3D structures to build ensemble models for nucleic acid motifs.

Keywords: DNA damage; DNA dynamics; DNA recognition; mutation; replication error.

Figure 1.

Nucleic acid base pairs form dynamic ensembles, which include alternative, lowly-populated, and short-lived conformational states that drive key biological processes. (A) A-T and G-C Watson-Crick bps form Hoogsteen conformations through 180° flips of purine bases, which play roles in protein-DNA recognition, DNA damage induction, accommodation, and repair, and damage bypass during replication. (B) G-T wobble mismatches form Watson-Crick-like conformations through tautomerization and ionization of the bases, while A-G mismatches form Watson-Crick-like conformation through 180° flips of purine bases to form Hoogsteen conformations. These Watson-Crick-like conformations facilitate nucleotide misincorporation and replicative errors. Modifications such as 8OG can alter the pre-existing conformational equilibria. (C) Comparison between the measured versus predicted DNA replicative fidelity for various DNA polymerases, mismatches, and DNA modifications: a, G-T misincorporation by AMV RT (Avian Myeloblastosis Virus reverse transcriptase) at pH 6.9; b, G-T misincorporation by AMV RT at pH 8.0; c, G-T misincorporation by AMV RT at pH 8.3; d, G-5BrU (5-bromouracil) misincorporation by AMV RT at pH 6.9; e, G-5BrU misincorporation by AMV RT at pH 8.4; f, G-T misincorporation by DNA pol ε at pH 7.4; g, G-T misincorporation by T7 pol at pH 7.4; h, G-T misincorporation by DNA pol β at pH 7.4; i, A-G misincorporation by DNA pol β at pH 7.4; j, A-G misincorporation by DNA pol β at pH 8.4; k, A-8OG misincorporation by DNA pol β at pH 7.4. The polymerase fidelities for each condition were measured using pre-steady state kinetic assay or simulated using kinetic modeling as described previously [1, 2]. (D) Mismatches or damaged nucleotides can adopt extra-helical conformations during damage repair. (E) Mismatches can bias the conformational ensemble toward structures recognized by proteins, increasing their binding affinity and resulting in mutation through competition with mismatch repair.

Figure 2.

Time diagram showing key developments in the conformational characterization of the A-C mismatch in duplex DNA. Some references are also made to foundational RNA studies.

Figure 3.

pH and sequence-dependent ensemble and energetics of the A-C mismatch. (A) The pH-dependent equilibrium of the A-C mismatch. The protonated wobble conformation is in dynamic equilibrium with a neutral species comprising two conformational states, the wobble and inverted wobble. (B) Histogram showing the difference in duplex stability (ΔΔG, kcal/mol) between A-C and A-T/U for DNA (blue) [3] and RNA (red) [4] at varying pH conditions. (C) Histogram of reported pKa values for A-C in duplex DNA (blue) [–8], duplex RNA (red) [6, 9, 10], and RNA loops (green) [–16]. No pKa value was reported for the A-C in the AAC, AA(T/U), and (T/U)AC contexts: n.d. indicates no data.

Figure 4.

Watson-Crick-like tautomeric A-C conformations. (A) Comparison of canonical A-T Watson-Crick bp, the protonated wobble conformation of A-C mismatch, and the Watson-Crick-like A-C tautomers. The wobble deviates from the Watson-Crick geometry indicated by the rectangle. (B) N4-methoxycytosine (mo4C) promotes Watson-Crick-like A-C tautomer and replicative errors by biasing cytosine to its imino-tautomeric form. (C) Crystal structures show that the A-C mismatch forms a wobble conformation when DNA polymerase adopts the inactive ajar conformation, whereas it forms a Watson-Crick-like conformation when the DNA polymerase adopts the catalytically active closed conformation. (D) Comparison of the C1’-C1’ distance and the valence angles subtended by the C1’-C1’ vector and glycosidic bonds of the purine (θ1) and pyrimidine (θ2) residues for base pairs within the insertion site of bacterial DNA pol I [17, 18]. DNA pol in complex with A-TTP, G-CTP, and Watson-Crick-like A-CTP at its insertion site forms the catalytically active closed conformation, whereas it forms an inactive ajar conformation when in complex with the A-CTP wobble.

Figure 5.

Other high-energy A-C conformations. (A) The reverse wobble A-C in which the entire adenine (or cytosine) nucleotide flips through the rearrangement of the backbone. (B) The reverse wobble-like A(syn)-C is like the reverse wobble but in which the sugar moiety of the adenine residue flips from the anti to syn conformation. (C) The 1,N6-ethenoadenine (εA) lesion promotes the A-C Hoogsteen conformation by disrupting the Watson-Crick face of the adenine base.

Figure 6.

Hypothetical free energy landscape of the A-C mismatch ensemble and how it is redistributed by changing the pH (in blue) and chemical modifications such as N4-methoxylation of cytosine (in red).