The DNA minidumbbell (MDB) is a recently identified non-B structure. The reported MDBs contain two TTTA, CCTG, or CTTG type II loops. At present, the knowledge and understanding of the sequence criteria for MDB formation are still limited. In this study, we performed a systematic high-resolution nuclear magnetic resonance (NMR) and native gel study to investigate the effect of sequence variations in tandem repeats on the formation of MDBs. Our NMR results reveal the importance of hydrogen bonds, base–base stacking, and hydrophobic interactions from each of the participating residues. We conclude that in the MDBs formed by tandem repeats, C–G loop-closing base pairs are more stabilizing than T–A loop-closing base pairs, and thymine residues in both the second and third loop positions are more stabilizing than cytosine residues. The results from this study enrich our knowledge on the sequence criteria for the formation of MDBs, paving a path for better exploring their potential roles in biological systems and DNA nanotechnology.

Quasi-loops, wherein the backbone has a discontinuous site in the loop, have been found to provide structural flexibility in the formation of DNA three-way junctions. Recently, a highly compact mini-dumbbell (MDB) structure composed of two adjacent CCTG or TTTA type II loops has been reported. Yet, it remains elusive if the presence of a quasi-loop will also facilitate the formation of an MDB. In this study, the possibility of whether an MDB can be formed containing a quasi-type II loop has been investigated. We first demonstrate that two adjacent CTTG type II loops can also form an MDB, which is thermodynamically more stable than those formed by CCTG and TTTA loops. Then, we systematically introduce quasi-type II loops with their discontinuous sites at different backbone positions in the CTTG MDB. Our results show that an MDB can be formed with a quasi-type II loop, in which there is a discontinuous site between the third thymine and the fourth guanine loop residues. The possible inclusion of a quasi-loop in MDBs expands the sequence criteria for the formation of MDBs by natural DNA sequences.

The non-B DNA structures formed by short tandem repeats on the nascent strand during DNA replication have been proposed to be the structural intermediates that lead to repeat expansion mutations. Tetranucleotide TTTA and CCTG repeat expansions have been known to cause reduction in biofilm formation in Staphylococcus aureus and myotonic dystrophy type 2 in human, respectively. In this study, we report the first three-dimensional minidumbbell (MDB) structure formed by natural DNA sequences containing two TTTA or CCTG repeats. The formation of MDB provides possible pathways for strand slippage to occur, which ultimately leads to repair escape and thus expansion mutations. Our result here shows that MDB is a highly compact structure composed of two type II loops. In addition to the typical stabilizing interactions in type II loops, MDB shows extensive stabilizing forces between the two loops, including two distinctive modes of interactions between the minor groove residues. The formation of MDB enriches the structural diversity of natural DNA sequences, reveals the importance of loop–loop interactions in unusual DNA structures, and provides insights into novel mechanistic pathways of DNA repeat expansion mutations.

Strand slippage has been found to occur in primer–templates containing a templating thymine, cytosine, and guanine, leading to the formation of misaligned structures with a single-nucleotide bulge. If remained in the active site of low-fidelity polymerases during DNA replication, these misaligned structures can ultimately bring about deletion mutations. In this study, we performed NMR investigations on primer–template models containing a templating adenine. Similar to our previous results on guanine, adenine templates are also less prone to strand slippage than pyrimidine templates. Misalignment occurs only in primer–templates that form a terminal C·G or G·C base pair. Together with our previous findings on thymine, cytosine, and guanine templates, the present study reveals strand slippage can occur in any kind of natural templating bases during DNA replication, providing insights into the origin of mutation hotspots in natural DNA sequences. In addition to the type of incoming base upon misincorporation, the propensity of strand slippage in primer–templates depends also on the type of templating base, its upstream and downstream bases.

An abasic site is the most common lesion in DNA. It is also an intermediate product formed during base excision repair. Previously, we demonstrated that strand slippage can occur in primer-template model systems containing any kind of natural templating bases, suggesting deletion and expansion errors are possible in any kind of sequences during DNA replication. In this study, nuclear magnetic resonance spectroscopic investigations have been performed to study the intrinsic effect of a templating abasic residue on strand slippage in primer-template models. A DNA hairpin model system containing an abasic site and a 5′-overhang region was used to mimic the situation that a dNTP has just been incorporated opposite the abasic site. Our results show that, after dNTP incorporation, strand slippage occurs regardless of the type of terminal base pair formed. Compared to natural templating bases, abasic sites possess a higher slippage propensity, implicating a higher chance of expansion or deletion errors during DNA replication.

Misaligned structures can result from strand slippage during DNA replication and, if not repaired, would lead to mutations. Previously, we showed that strand slippage can occur upon misincorporation of a dNTP opposite thymine and cytosine templates, resulting in a misaligned structure with a T- or C-bulge. The formation propensity for misaligned structures was found to depend on the type of terminal base pair. In this study, we performed NMR investigations on primer-template models containing a guanine template. Our results reveal guanine templates are less prone to strand slippage than pyrimidine templates. Misalignment was found to occur only in 5′-CG templates with a downstream purine. In addition to the significance of terminal base pair and upstream nucleotide, the present study reveals the importance of the templating base and its downstream nucleotide, which also determine the propensity of strand slippage in primer-templates.

Our previous studies have shown that misaligned structures can occur upon misincorporation of a dNTP opposite thymine templates. The formation of misaligned structures during DNA replication, if not repaired properly, can be bypassed and extended by low-fidelity polymerases and ultimately lead to mutations. In this study, the base pair structures at the replicating sites of a set of primer−template models which mimic the situation upon misincorporation of a dNTP opposite cytosine templates have been determined. High-resolution NMR structural results show that misaligned structures with a C-bulge can be formed upon incorporation of dCTP, dTTP, and dATP opposite 5′-GC, 5′-AC, and 5′-TC templates, respectively. The stabilities of misaligned structures depend on the types of terminal base pairs at the replicating sites. Together with the structural findings in thymine templates, we conclude that terminal G·C and C·G base pairs always contribute a larger stabilizing effect to the misaligned structures containing a pyrimidine bulge than terminal A·T and T·A base pairs. Misalignment and thus deletion mutation are more likely to occur if misincorporation of a nucleotide opposite a pyrimidine template can cause template slippage to form a terminal G·C or C·G base pair. Although misalignment also occurs when the newly formed terminal base pair is an A·T base pair or a T·A base pair, both misaligned and mismatched conformers coexist, which can lead to deletion and substitution mutations, respectively.

•A DNA sequence containing two CCTG repeats can form a mini-dumbbell structure.•The mini-dumbbell provides a possible pathway for two-repeat expansion.•Fast exchange between two competing dumbbells results in a mini-loop structure.•The mini-loop provides a possible pathway for one-repeat expansion.•Co-existence of these structures leads to three or larger size repeat expansions.Tetranucleotide CCTG repeat expansion is associated with myotonic dystrophy type 2, which is an inherited and progressive muscle degeneration disease. Yet, no cure is available and the molecular mechanism of repeat expansion remains elusive. In this study, we used high-resolution nuclear magnetic resonance spectroscopy to reveal a mini-dumbbell structure formed by two CCTG repeats. Upon slippage in the nascent strand during DNA replication, the formation of the mini-dumbbell provides a possible pathway for a two-repeat expansion. In addition, fast exchange between two competing mini-dumbbells among three repeats results in a mini-loop structure that accounts for one-repeat expansion. These mini-dumbbell and mini-loop intermediates can also co-exist at multiple sites in CCTG repeats, leading to three or larger size repeat expansions.

1-Methyladenine (m1A) alters T·A Watson–Crick to T·m1A Hoogsteen base pair. Owing to its conversion to N6-methyladenine (m6A) at higher temperatures, thermodynamic studies of m1A-containing DNAs using conventional melting methods are subject to the influence of m6A species. In this study, we applied nuclear magnetic resonance spectroscopy to determine the base pairing modes and effect of m1A on thermodynamic stability of double-helical DNA. The observed base pairing modes account for the destabilizing trend which follows the order T·m1A ∼ G·m1A < A·m1A < C·m1A, providing insights into the m1A flipping process and enhancing our understandings of the mutagenicity of m1A.

Oxidation of guanine in DNA can lead to mutagenic lesions such as 7-hydro-8-oxoguanine (oG). Upon further oxidation, a more mutagenic lesion, spirominodihydantoin (Sp), can occur. In this study, nuclear magnetic resonance (NMR) investigations were performed to determine the structural features of DNA primer–template models with 5′-GG, 5′-G(oG), 5′-G(Sp) and 5′-T(Sp) templates, that mimic the situation in which the downstream G of the template has been oxidized to oG or hyperoxidized to Sp. Our results show that misalignment occurs only in the 5′-G(Sp) and 5′-T(Sp) templates, providing structural insights into the observed differences in mutagenicity of Sp and oG during DNA replication.

•TTTA repeat expansions affect the biofilm matrix of Staphylococcus aureus.•A sequence containing three TTTA repeats can form two dumbbell conformers.•Fast exchange of the two conformers results in an averaged 2-nt mini-loop structure.•The occurrence of these structures in nascent strands can lead to repeat expansion.•The mini-loop and dumbbell account for one and two repeat expansions, respectively.One and two TTTA repeat expansions have been found in the coding region of icaC gene of Staphylococcus aureus variants which influence the expression of IcaC protein and alter the phenotype. Yet, the mechanism of these small-size TTTA repeat expansions remains unclear. In this study, we performed high-resolution nuclear magnetic resonance spectroscopic studies on TTTA repeats. Our results show that a DNA sequence containing three TTTA repeats can fold into dumbbell structures with a 3′ or 5′-overhang. Exchange of these dumbbells makes the sequence behave like a 2-nt TT mini-loop at 25 °C. The occurrence of these mini-loop and dumbbell structures in the nascent strand during DNA replication provides possible mechanistic pathways which account for one and two repeat expansions.

It has long been recognized that T·T mismatches can adopt two different modes of exchangeable wobble base pairs in which no preferential pairing mode has been observed. In this study, we have performed a systematic nuclear magnetic resonance (NMR) investigation to study the sequence context effect on the pairing modes of T·T mismatches. Our results reveal for the first time that preferential pairing mode does exist in T·T mismatches with specific type of flanking base pairs.Highlights► We study the sequence context effect on the pairing modes of T·T mismatches. ► We demonstrate the presence of preferential pairing mode in T·T mismatches. ► The pairing mode with flanking purines differs from that with flanking pyrimidines. ► These different pairing modes usually undergo exchange at room temperature.

Methylation at the N1 site of adenine leads to the formation of cytotoxic 1-methyladenine (m1A). Since the N1 site of adenine is involved in the hydrogen bonding of T·A and A·T Watson–Crick base pairs, it is expected that the pairing interactions will be disrupted upon 1-methylation. In this study, high-resolution NMR investigations were performed to determine the effect of m1A on double-helical DNA structures. Interestingly, instead of disrupting hydrogen bonding, we found that 1-methylation altered the T·A Watson–Crick base pair to T(anti)·m1A(syn) Hoogsteen base pair, providing insights into the observed differences in AlkB-repair efficiency between dsDNA and ssDNA.