DNA Bulge Research Articles

Deciphering HMGB1: Across a spectrum of DNA and nucleosome dynamics

AbstractHMGB1 is the most abundant nonhistone nuclear protein, which has been widely studied for its roles in the cytoplasm as an autophagy mediator and in the extracellular matrix as an inflammatory molecule. Studies concerning HMGB1's actual role and its binding within the nucleus are inadequate. Through this in vitro study, we aimed to discern the binding parameters of HMGB1 with various types of DNA, nucleosomes, and chromatin. HMGB1 binds differentially to different DNA, with a high affinity for altered DNA structures such as triplex and bulge DNA. Remodelling of nucleosome by CHD7 remodeller was negatively impacted by the binding of HMGB1. We also found that HMGB1 binds to the linker DNA of chromatin. Findings from this study shed light on the diverse roles HMGB1 may play in transcription, gene expression, viral replication, CHARGE syndrome and so forth.

A Single Interfacial Point Mutation Rescues Solution Structure Determination of the Complex of HMG-D with a DNA Bulge.

Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.

DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL−1, 1.5 copies⋅μL−1, and 0.17 copies⋅μL−1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

Aberrant repair of 8-oxoguanine in short DNA bulges

The presence of DNA damage can increase the likelihood of DNA replication errors and promote mutations. In particular, pauses of DNA polymerase at the site of damage can lead to polymerase slippage and the formation of 1–2 nucleotide bulges. Repair of such structures using an undamaged DNA template leads to small deletions. One of the most abundant oxidative DNA lesions, 8-oxoguanine (oxoG), has been shown to induce small deletions but the mechanism of this phenomenon is currently unknown. We have studied the aberrant repair of oxoG, located in one- and two-nucleotide bulges, by the Escherichia coli and human base excision repair systems. Our results indicate that the repair in such substrates can serve as a mechanism for fixing small deletions in bacteria but not in humans.

Aberrant Repair of 8-Oxoguanine in Short DNA Bulges.

The presence of DNA damage can increase the likelihood of DNA replication errors and promote mutations. In particular, pauses of DNA polymerase at the site of damage can lead to polymerase slippage and the formation of 1-2-nucleotide bulges. Repair of such structures using an undamaged DNA template leads to small deletions. One of the most abundant oxidative DNA lesions, 8-oxoguanine (oxoG), was shown to induce small deletions, but the mechanism of this phenomenon is currently unknown. We studied the aberrant repair of oxoG located in one- and two-nucleotide bulges by the Escherichia coli and human base excision repair systems. Our results indicate that the repair in such substrates can serve as a mechanism for fixing small deletions in bacteria but not in humans.

Flexibility of telomerase in binding the RNA template and DNA telomeric repeat

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.

Differential repair enzyme-substrate selection within dynamic DNA energy landscapes.

We demonstrate that reshaping of the dynamic, bulged-loop energy landscape of DNA triplet repeat ensembles by the presence of an abasic site alters repair outcomes by the APE1 enzyme. This phenomenon depends on the structural context of the lesion, despite the abasic site always having the same neighbors in sequence space. We employ this lesion-induced redistribution of DNA states and a kinetic trap to monitor different occupancies of the DNA bulge loop states. We show how such dynamic redistribution and associated differential occupancies of DNA states impact APE1 repair outcomes and APE1 induced interconversions. We correlate the differential biophysical properties of the dynamic, DNA ensemble states, with their ability to be recognized and processed as substrates by the APE1 DNA repair enzyme. Enzymatic digestions and biophysical characterizations reveal that APE1 cuts a fraction (10-12%) of the dynamic, rollameric substrates within the initial kinetic distribution. APE1 interactions also 'induce' rollamer redistribution from a kinetically trapped distribution to an equilibrium distribution, the latter not containing viable APE1 substrates. We distinguish between kinetically controlled ensemble (re)distributions of potential DNA substrates, versus thermodynamically controlled ensemble (re)distribution; features of importance to DNA regulation. We conclude that APE1 activity catalyzes/induces ensembles that represent the thermodynamically optimal loop distribution, yet which also are nonviable substrate states for abasic site cleavage by APE1. We propose that by inducing substrate redistributions in a dynamic energy landscape, the enzyme actually reduces the available substrate competent species for it to process, reflective of a regulatory mechanism for enzymatic self-repression. If this is a general phenomenon, such a consequence would have a profound impact on slowing down and/or misdirecting DNA repair within dynamic energy landscapes, as exemplified here within triplet repeat domains. In short, APE1-instigated redistribution of potential substrates induces a preferred pathway to an equilibrium ensemble of enzymatically incompetent states.

Ab initio multi-level layered elongation method and its application to local interaction analysis between DNA bulge and ligand molecules.

A multi-level layered elongation method was developed for efficiently analyzing the electronic states of local structures in large bio/nano-systems at the full ab initio level of theory. The original elongation method developed during the last three decades in our group has focused on the system in one direction from one terminal to the other terminal to sequentially construct the electronic states of a polymer, called a theoretical synthesis of polymers. In this study, an important region termed the central (C) part is targeted in a large polymer and the remainder are terminal (T) parts. The electronic structures along with polymer elongation are calculated repeatedly from both end T parts to the C central part at the same time. The important C part is treated with large basis sets (high level) and the other regions are treated with small basis sets (low level) in the ab initio theoretical framework. The electronic structures besides the C part can be reused for other systems with different structures at the C part, which renders the method computationally efficient. This multi-level layered elongation method was applied to the investigation on DNA single bulge recognition of small molecules (ligands). The reliability and validity of our approach were examined in comparison with the results obtained by direct calculations using a conventional quantum chemical method for the entire system. Furthermore, stabilization energies by the formation of the complex of bulge DNA and a ligand were estimated with basis set superposition error corrections incorporated into the elongation method.

Predicting stability of DNA bulge at mononucleotide microsatellite

Mononucleotide microsatellites are clinically and forensically crucial DNA sequences due to their high mutability and abundance in the human genome. As a mutagenic intermediate of an indel in a microsatellite and a consequence of probe hybridization after such mutagenesis, a bulge with structural degeneracy sliding within a microsatellite is formed. Stability of such dynamic bulges, however, is still poorly understood despite their critical role in cancer genomics and neurological disease studies. In this paper, we have built a model that predicts the thermodynamics of a sliding bulge at a microsatellite. We first identified 40 common bulge states that can be assembled into any sliding bulges, and then characterized them with toehold exchange energy measurement and the partition function. Our model, which is the first to predict the free energy of sliding bulges with more than three repeats, can infer the stability penalty of a sliding bulge of any sequence and length with a median prediction error of 0.22 kcal/mol. Patterns from the prediction clearly explain landscapes of microsatellites observed in the literature, such as higher mutation rates of longer microsatellites and C/G microsatellites.

RNA Editing of the Human DNA Glycosylase NEIL1 Alters Its Removal of 5-Hydroxyuracil Lesions in DNA.

Editing of the pre-mRNA of the DNA repair glycosylase NEIL1 results in substitution of a Lys with Arg in the lesion recognition loop of the enzyme. Unedited (UE, Lys242) NEIL1 removes thymine glycol lesions in DNA ∼30 times faster than edited (Ed, Arg242) NEIL1. Herein, we evaluated recognition and excision mediated by UE and Ed NEIL1 of 5-hydroxyuracil (5-OHU), a highly mutagenic lesion formed via oxidation of cytosine. Both NEIL1 isoforms catalyzed low levels of 5-OHU excision in single-stranded DNA, bubble and bulge DNA contexts and in duplex DNA base paired with A. Removal of 5-OHU in base pairs with G, T, and C was found to be faster and proceed to a higher overall extent with UE than with Ed NEIL1. In addition, the presence of mismatches adjacent to 5-OHU magnified the hampered activity of the Ed isoform. However, Ed NEIL1 was found to exhibit higher affinity for 5-OHU:G and 5-OHU:C duplexes than UE NEIL1. These results suggest that NEIL1 plays an important role in detecting and capturing 5-OHU lesions in inappropriate contexts, in a manner that does not lead to excision, to prevent mutations and strand breaks. Indeed, inefficient removal of 5-OHU by NEIL1 from 5-OHU:A base pairs formed during replication would thwart mutagenesis. Notably, nonproductive engagement of 5-OHU by Ed NEIL1 suggests the extent of 5-OHU repair will be reduced under cellular conditions, such as inflammation, that increase the extent of NEIL1 RNA editing. Tipping the balance between the two NEIL1 isoforms may be a significant factor leading to genome instability.

Lesion Recognition and Cleavage of Damage-Containing Quadruplexes and Bulged Structures by DNA Glycosylases.

Human telomeres as well as more than 40% of human genes near the promoter regions have been found to contain the sequence that may form a G-quadruplex structure. Other non-canonical DNA structures comprising bulges, hairpins, or bubbles may have a functionally important role during transcription, replication, or recombination. The guanine-rich regions of DNA are hotspots of oxidation that forms 7,8-dihydro-8-oxoguanine, thymine glycol, and abasic sites: the lesions that are handled by the base excision repair pathway. Nonetheless, the features of DNA repair processes in non-canonical DNA structures are still poorly understood. Therefore, in this work, a comparative analysis of the efficiency of the removal of a damaged nucleotide from various G-quadruplexes and bulged structures was performed using endonuclease VIII-like 1 (NEIL1), human 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III (NTH1), and prokaryotic formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease VIII (Nei). All the tested enzymes were able to cleave damage-containing bulged DNA structures, indicating their important role in the repair process when single-stranded DNA and intermediate non–B-form structures such as bubbles and bulges are formed. Nevertheless, our results suggest that the ability to cleave damaged quadruplexes is an intrinsic feature of members of the H2tH structural family, suggesting that these enzymes can participate in the modulation of processes controlled by the formation of quadruplex structures in genomic DNA.

Cationic FeII Triplex-Forming Metallohelices as DNA Bulge Binders.

Bulges are essential structural elements in nucleic acids. The detection and targeting of bulged DNA sequences are highly important. Small molecules capable of targeting DNA bulges have attracted considerable attention because they cannot only be used as reagents for bulge recognition, but also as potential therapeutic drugs. Herein, the interactions of DNA duplexes, containing bulges of various sizes and base compositions, with a series of FeII triplex-forming metallohelices are reported. The results obtained, with the aid of molecular biophysics methods, show that the investigated metallohelices prefer to bind to bulged DNA, rather than double-stranded DNA, and that their binding affinities towards bulges differ among individual metallohelices. Moreover, their binding affinities towards bulges strongly depend on the bulge size and the base composition of the bulge loop. The investigated metallohelices can enter eukaryotic cells and accumulate in the cell nucleus, allowing them to interact with nucleic acids. Hence, it is reasonable to suggest that the interaction of metallohelices with nucleic acid bulges might contribute to the mechanism of their biological activity.

Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA

N6-methyladenine (N6-mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N6-mA undergo drastic fluctuation during early embryogenesis, indicative of active regulation. Here we show that the 2-oxoglutarate-dependent oxygenase ALKBH1 functions as a nuclear eraser of N6-mA in unpairing regions (e.g., SIDD, Stress-Induced DNA Double Helix Destabilization regions) of mammalian genomes. Enzymatic profiling studies revealed that ALKBH1 prefers bubbled or bulged DNAs as substrate, instead of single-stranded (ss-) or double-stranded (ds-) DNAs. Structural studies of ALKBH1 revealed an unexpected “stretch-out” conformation of its “Flip1” motif, a conserved element that usually bends over catalytic center to facilitate substrate base flipping in other DNA demethylases. Thus, lack of a bending “Flip1” explains the observed preference of ALKBH1 for unpairing substrates, in which the flipped N6-mA is primed for catalysis. Co-crystal structural studies of ALKBH1 bound to a 21-mer bulged DNA explained the need of both flanking duplexes and a flipped base for recognition and catalysis. Key elements (e.g., an ALKBH1-specific α1 helix) as well as residues contributing to structural integrity and catalytic activity were validated by structure-based mutagenesis studies. Furthermore, ssDNA-seq and DIP-seq analyses revealed significant co-occurrence of base unpairing regions with N6-mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N6-mA turnover of unpairing regions associated with dynamic chromosome regulation.

Evaluation of cytotoxic potential of structurally well-characterized RNA targeted ionic non-steroidal anti-inflammatory (NSAID) Cu(ii) & Zn(ii) DACH-mefenamato drug conjugates against human cancer cell lines.

New RNA targeted ionic [Cu(DACH)2(H2O)2](mef)2, 1 and [Zn(DACH)2(H2O)2](mef)2, 2 drug conjugates were synthesized and characterized by spectroscopic techniques FT-IR, UV-vis, EPR in case of 1 and 1H and 13C NMR in case of 2, ESI-MS, thermogravimetric analysis and single-crystal X-ray structure determination in case of 1. The interaction studies of 1 & 2 with most likely drug targets like ctDNA and tRNA were performed which demonstrated that the complexes 1 and 2 exhibited strong preferential binding to tRNA as compared to ctDNA, Kb = 2.52(±0.04) × 105 M−1, 7.85(±0.02) × 104 M−1, respectively. Scanning electron microscopy analyses of complex-ctDNA/tRNA condensates suggested the interaction of complexes with ctDNA/tRNA had occurred, followed by lengthening of DNA double helix and bulge region of tRNA. Cytotoxic activity of 1 and 2 against human cancer cell lines namely; MCF-7 (breast), HeLa (cervical), MIA-PA-CA 2 (pancreatic), A-498 (kidney), Hep-G2 (hepatoma) was evaluated by SRB assay. The obtained results showed that copper complex 1 was an outstanding cytotoxic agent with remarkably good GI50 value (<10 μg ml−1) against the tested cancer cell lines except for MIA-PA-CA 2, while zinc complex 2 revealed moderate cytotoxicity against all the tested cancer cell lines.

Stabilization of DNA Loop Structures by Large Cations.

The DNA-binding properties of large cations differ from those of metal ions due to steric exclusion from base-paired regions. In this study, the thermal stability of DNA secondary structures, including duplexes, internal loops, bulge loops, hairpin loops, dangling ends, and G-quadruplexes, was investigated in the presence of cations of different sizes. Large cations, such as tetrabutylammonium and tetrapentylammonium ions, reduced the stability of fully matched duplexes but increased the stability of duplexes with a long loop. The cations also increased the stability of G-quadruplexes with a long loop, and the degree of stabilization was greater for low-stability G-quadruplexes. Analysis of the salt concentration dependence indicates that large cations bind to the loop nucleotides, leading to counteracting the destabilization effect on base pairing. It is likely that binding occurs when loop nucleotides are sufficiently flexible to allow for greater accessibility for large cations. These results provide insight into nucleic acid interactions with large cationic molecules and suggest a potential method for stabilizing noncanonical DNA structures under intracellular conditions.

Mechanism of DNA translocation underlying chromatin remodelling by Snf2.

Chromatin remodellers include diverse enzymes with distinct biological functions, but nucleosome-sliding activity appears to be a common theme1,2. Among the remodelling enzymes, Snf2 serves as the prototype to study the action of this protein family. Snf2 and related enzymes share two conserved RecA-like lobes3, which by themselves are able to couple ATP hydrolysis to chromatin remodelling. The mechanism by which these enzymes couple ATP hydrolysis to translocate the nucleosome along the DNA remains unclear2,4-8. Here we report the structures of Saccharomyces cerevisiae Snf2 bound to the nucleosome in the presence of ADP and ADP-BeFx. Snf2 in the ADP-bound state adopts an open conformation similar to that in the apo state, and induces a one-base-pair DNA bulge at superhelix location 2 (SHL2), with the tracking strand showing greater distortion than the guide strand. The DNA distortion propagates to the proximal end, leading to staggered translocation of the two strands. The binding of ADP-BeFx triggers a closed conformation of the enzyme, resetting the nucleosome to a relaxed state. Snf2 shows altered interactions with the DNA in different nucleotide states, providing the structural basis for DNA translocation. Together, our findings suggest a fundamental mechanism for the DNA translocation that underlies chromatin remodelling.

A twist defect mechanism for ATP-dependent translocation of nucleosomal DNA.

As superfamily 2 (SF2)-type translocases, chromatin remodelers are expected to use an inchworm-type mechanism to walk along DNA. Yet how they move DNA around the histone core has not been clear. Here we show that a remodeler ATPase motor can shift large segments of DNA by changing the twist and length of nucleosomal DNA at superhelix location 2 (SHL2). Using canonical and variant 601 nucleosomes, we find that the Saccharomyces cerevisiae Chd1 remodeler decreased DNA twist at SHL2 in nucleotide-free and ADP-bound states, and increased twist with transition state analogs. These differences in DNA twist allow the open state of the ATPase to pull in ~1 base pair (bp) by stabilizing a small DNA bulge, and closure of the ATPase to shift the DNA bulge toward the dyad. We propose that such formation and elimination of twist defects underlie the mechanism of nucleosome sliding by CHD-, ISWI-, and SWI/SNF-type remodelers.

Bulge oligonucleotide as an inhibitory agent of bacterial topoisomerase I

Bacterial topoisomerase I (Btopo I) was defined as potential target for discovery of new antibacterial compounds. Various oligonucleotides containing bulge structure were designed and synthesised as inhibitors to Btopo I in this investigation. The results of this study demonstrated that the designed oligonucleotides display high inhibitory efficiency on the activity of Btopo I and the inhibitory effect could be modulated by the amount of bulge DNA bases. The most efficient one among them showed an IC50 value of 63.1 nM in its inhibition on the activity of Btopo I. In addition, our studies confirmed that the designed oligonucleotide would induce irreversible damages to Btopo I and without any effects occur to eukaryotic topoisomerase I. It is our hope that the results provided in these studies could provide a novel way to inhibit Btopo I.

Trifunctional fluorescent unnatural nucleoside: Label free detection of T-T/C-C base mismatches, abasic site and bulge DNA

The detection and targeting of both the mismatched and abasic DNA is highly important which would ultimately help in designing new diagnostics and chemotherapeutics. Furthermore, sensing and targeting the bulge sequence with a fluorescent probe would be useful to study the role of bulges in nucleic acid function or could have significant therapeutic potential. Thus, detection of specific bulges by small fluorescent molecules is an attractive research area since the past several years. Many attempts have been made to prepare such compounds. We report herein a label free strategy for the detection of pyrimidine base mismatches (T/T and C/C), sensing of abasic site, and pyrimidine base bulge DNA using an unnatural tetrazolylpyrene nucleoside (TPyBDo) as a bare fluorescent probe. The H-bonding/hydrophobic force mediated interactions allow the sensing of all three deformed DNA via an enhancement of fluorescence signal using our simple “Just-Mix and Read” strategy. The binding of the probe to all the three deformed DNA duplexes is accompanied by an increase in the thermal melting stability of the deformed DNAs. That the probe binds efficiently to the minor groove near the deformed site was evident from spectroscopic studies. All the spectral evidences open up a multitude of possibilities for using our probe, tetrazolylpyrene nucleoside, as an efficient fluorescent light-up bio-probe for label free DNA detection.

A 2,7-diamino-1,4,8-triazanaphthalene derivative selectively binds to cytosine bulge DNA only at a weakly acidic pH

The synthesis and properties of 2,7-diamino-1,4,8-triazanaphthalene (azaDANP) are described. AzaDANP is protonated only at a weakly acidic pH to bind to the cytosine bulge DNA duplex selectively. Upon binding of azaDANP to the cytosine bulge DNA, a new absorption band at 407 nm appears, and the absorption change of azaDANP on binding to the target is very sensitive to environmental pH with a bell-shaped pH-absorption profile.