-
Biophysical Journal Nov 2022DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied...
DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here, we investigate the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We perform coarse-grained molecular dynamics simulations and confirm the results with nanoindentation experiments using atomic force microscopy. We observe various deformation modes, including untension, linear elasticity, duplex dissociation, and single-stranded component stretch. We find that the mechanical properties of a DNA architecture are correlated with those of its components. However, the structure shows complex behaviors which may not be predicted by components alone and the architectural design must be considered.
Topics: DNA; Nanostructures; Microscopy, Atomic Force; Molecular Dynamics Simulation; Elasticity; Nucleic Acid Conformation
PubMed: 36181269
DOI: 10.1016/j.bpj.2022.09.036 -
BMC Genomics Aug 2022DNA and RNA sequencing are widely used techniques to investigate genomic modifications and gene expression. The costs for sequencing dropped dramatically in the last...
DNA and RNA sequencing are widely used techniques to investigate genomic modifications and gene expression. The costs for sequencing dropped dramatically in the last decade. However, due to material and labor intense steps, the sample preparation costs could not keep up with that pace. About 80% of the total costs occur prior to sequencing during DNA/RNA extraction, enrichment steps and subsequent library preparation. In this study, we investigate the potential of pooling different organisms samples prior to DNA/RNA extraction to significantly reduce costs in preparative steps. Similar to the common procedure of ligated DNA tags to pool (c)DNA samples, sequence diversity of different organisms intrinsically provide unique sequences that allow separation of reads after sequencing. With this approach, sample pooling can occur before DNA/RNA isolation and library preparation. We show that pooled sequencing of three related bacterial organisms is possible without loss of data quality at a cost reduction of approx. 50% in DNA- and RNA-seq approaches. Furthermore, we show that this approach is highly efficient down to the level of a shared genus and is, therefore, widely applicable in sequencing facilities and companies with diverse sample pools.
Topics: DNA; High-Throughput Nucleotide Sequencing; Metagenome; Metagenomics; RNA; Sequence Analysis, DNA; Sequence Analysis, RNA
PubMed: 35999507
DOI: 10.1186/s12864-022-08831-y -
Journal of Radiation Research Sep 2017Production of reactive oxygen and nitrogen species (ROS), such as hydrogen peroxide, superoxide and hydroxyl radicals, has been linked to cancer, and these oxidative...
Production of reactive oxygen and nitrogen species (ROS), such as hydrogen peroxide, superoxide and hydroxyl radicals, has been linked to cancer, and these oxidative molecules can damage DNA. Base excision repair (BER), a major repair system maintaining genome stability over a lifespan, has an important role in repairing oxidatively induced DNA damage. Failure of BER leads to toxic consequences in ROS-exposed cells, and ultimately can contribute to the pathobiology of disease. In our previous report, we demonstrated that oxidized nucleotide insertion by DNA polymerase β (pol β) impairs BER due to ligation failure and leads to formation of a cytotoxic repair intermediate. Biochemical and cytotoxic effects of ligation failure could mediate genome stability and influence cancer therapeutics. In this review, we discuss the importance of coordination between pol β and DNA ligase I during BER, and how this could be a fundamental mechanism underlying human diseases such as cancer and neurodegeneration. A summary of this work was presented in a symposium at the International Congress of Radiation Research 2015 in Kyoto, Japan.
Topics: DNA; DNA Ligase ATP; DNA Polymerase beta; DNA Repair; Nucleotides; Oxidation-Reduction; Substrate Specificity; Templates, Genetic
PubMed: 28992331
DOI: 10.1093/jrr/rrx027 -
Environmental and Molecular Mutagenesis Apr 2024In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations,...
In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased ~two-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol β performs multiple insertions on G4 substrates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol β products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol β prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.
Topics: DNA Repair; Vascular Endothelial Growth Factor A; DNA, B-Form; Oxidative Stress; DNA; DNA Damage
PubMed: 37606505
DOI: 10.1002/em.22570 -
Advanced Science (Weinheim,... Nov 2023DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot...
DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot be actively processed in DNA pools. An active DNA data editing process is developed using splint ligation in a droplet-controlled fluidics (DCF) system. DNA fragments of discrete sizes (100-500 bps) are synthesized for droplet assembly, and programmed sequence information exchange occurred. The encoded DNA sequences are processed in series and parallel to synthesize the determined DNA pools, enabling random access using polymerase chain reaction amplification. The sequencing results of the assembled DNA data pools can be orderly aligned for decoding and have high fidelity through address primer scanning. Furthermore, eight 90 bps DNA pools with pixel information (png: 0.27-0.28 kB), encoded by codons, are synthesized to create eight 270 bps DNA pools with an animation movie chip file (mp4: 12 kB) in the DCF system.
Topics: DNA; Polymerase Chain Reaction
PubMed: 37755129
DOI: 10.1002/advs.202303197 -
Cold Spring Harbor Protocols May 2020High-throughput sequencing methods have created exciting opportunities to explore the regulatory landscape of the entire genome. Here we introduce methods to...
High-throughput sequencing methods have created exciting opportunities to explore the regulatory landscape of the entire genome. Here we introduce methods to characterize the genomic locations of bound proteins, open chromatin, and sites of DNA-DNA contact in embryos. These methods include chromatin immunoprecipitation followed by sequencing (ChIP-seq), a combination of DNase I digestion and sequencing (DNase-seq), the assay for transposase-accessible chromatin and sequencing (ATAC-seq), and the use of proximity-based DNA ligation followed by sequencing (Hi-C).
Topics: Animals; Chromatin; Chromatin Immunoprecipitation; DNA; Embryo, Nonmammalian; Genome; Genomics; High-Throughput Nucleotide Sequencing; Xenopus
PubMed: 32123020
DOI: 10.1101/pdb.top097915 -
Current Opinion in Genetics &... Dec 2021DNA double-strand breaks can be repaired through ligation-based pathways (non-homologous end-joining) or replication-based pathways (homologous recombination) in... (Review)
Review
DNA double-strand breaks can be repaired through ligation-based pathways (non-homologous end-joining) or replication-based pathways (homologous recombination) in eukaryotic cells. The decisions that govern these outcomes are widely viewed as a competition between factors that recognize DNA ends and physically promote association of factors specific to each pathway, commonly known as 'pathway choice'. Here I review recent results in the literature and propose that this decision is better described as a sequential set of binding and end processing events, with non-homologous end joining as the first decision point. Physical association and co-localization of end resection factors with non-homologous end-joining factors suggests that ends are transferred between these complexes, thus the ultimate outcome is not the result of a competition but is more akin to a relay race that is determined by the efficiency of the initial end-joining event and the availability of activated DNA end-processing enzymes.
Topics: Animals; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; Homologous Recombination; Mammals
PubMed: 34293662
DOI: 10.1016/j.gde.2021.06.011 -
DNA Repair Nov 2020DNA methylation on cytosine in CpG islands generates 5-methylcytosine (5mC), and further modification of 5mC can result in the oxidized variants 5-hydroxymethyl (5hmC),...
DNA methylation on cytosine in CpG islands generates 5-methylcytosine (5mC), and further modification of 5mC can result in the oxidized variants 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxy (5caC). Base excision repair (BER) is crucial for both genome maintenance and active DNA demethylation of modified cytosine products and involves substrate-product channeling from nucleotide insertion by DNA polymerase (pol) β to the subsequent ligation step. Here, we report that, in contrast to the pol β mismatch insertion products (dCTP, dATP, and dTTP), the nicked products after pol β dGTP insertion can be ligated by DNA ligase I or DNA ligase III/XRCC1 complex when a 5mC oxidation modification is present opposite in the template position in vitro. A Pol β K280A mutation, which perturbates the stabilization of these base modifications within the active site, hinders the BER ligases. Moreover, the nicked repair intermediates that mimic pol β mismatch insertion products, i.e., with 3'-preinserted dGMP or dTMP opposite templating 5hmC, 5fC or 5caC, can be efficiently ligated, whereas preinserted 3'-dAMP or dCMP mismatches result in failed ligation reactions. These findings herein contribute to our understanding of the insertion tendencies of pol β opposite different cytosine base forms, the ligation properties of DNA ligase I and DNA ligase III/XRCC1 complex in the context of gapped and nicked damage-containing repair intermediates, and the efficiency and fidelity of substrate channeling during the final steps of BER in situations involving oxidative 5mC base modifications in the template strand.
Topics: 5-Methylcytosine; CpG Islands; DNA; DNA Damage; DNA Ligase ATP; DNA Methylation; DNA Polymerase beta; DNA Repair; Humans; Poly-ADP-Ribose Binding Proteins; X-ray Repair Cross Complementing Protein 1
PubMed: 32853828
DOI: 10.1016/j.dnarep.2020.102945 -
DNA Repair Jan 20225',8-cyclo-2-deoxy nucleosides (cdPus) are the smallest tandem purine lesions including 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG). They...
5',8-cyclo-2-deoxy nucleosides (cdPus) are the smallest tandem purine lesions including 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG). They can inhibit DNA and RNA polymerases causing mutations, DNA strand breaks, and termination of DNA replication and gene transcription. cdPus can be removed by nucleotide excision repair with low efficiency allowing them to accumulate in the genome. Recent studies suggest that cdPus can be induced in damaged nucleotide pools and incorporated into the genome by DNA polymerases. However, it remains unknown if and how DNA polymerases can incorporate cdPus. In this study, we examined the incorporation of cdAs by human DNA repair polymerases, DNA polymerases β (pol β), and pol η during base excision repair. We then determined the efficiency of cdA incorporation by the polymerases using steady-state kinetics. We found that pol β and pol η incorporated cdAs opposite dT and dC with low efficiency, and incorporated cdAs were readily extended and ligated into duplex DNA. Using molecular docking analysis, we found that the 5',8-covalent bond in cdA disrupted its hydrogen bonding with a template base suggesting that the phosphodiester bond between the 3'-terminus nucleotide and the α-phosphate of cdATP were generated in the absence of hydrogen bonding. The enzyme kinetics analysis further suggests that pol β and pol η increased their substrate binding to facilitate the enzyme catalysis for cdA incorporation. Our study reveals unique mechanisms underlying the accumulation of cdPu lesions in the genome resulting from nucleotide incorporation by repair DNA polymerases.
Topics: DNA; DNA Polymerase beta; DNA Repair; DNA-Directed DNA Polymerase; Deoxyadenosines; Humans; Kinetics; Molecular Docking Simulation
PubMed: 34871863
DOI: 10.1016/j.dnarep.2021.103258 -
Scientific Reports May 2024Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of...
Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.
Topics: SARS-CoV-2; Software; Polymerase Chain Reaction; Oligonucleotides; COVID-19; Computational Biology; DNA, Single-Stranded
PubMed: 38750104
DOI: 10.1038/s41598-024-59497-3