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Methods in Molecular Biology (Clifton,... 2020Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any...
Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allow detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.
Topics: Apoptosis; Chromosomes; DNA; DNA Breaks, Double-Stranded; DNA Ligases; DNA Primers; DNA Repair; High-Throughput Nucleotide Sequencing; Histone-Lysine N-Methyltransferase; Humans; Myeloid-Lymphoid Leukemia Protein; Polymerase Chain Reaction; Translocation, Genetic; Workflow
PubMed: 31989561
DOI: 10.1007/978-1-0716-0223-2_15 -
Structure (London, England : 1993) Mar 2022DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there...
DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the physical basis for this regulation. Here, we use single-particle cryoelectron microscopy (cryo-EM) to analyze an archaeal DNA ligase and heterotrimeric PCNA in complex with a single-strand DNA break. The cryo-EM structures highlight a continuous DNA-binding surface formed between DNA ligase and PCNA that supports the distorted conformation of the DNA break undergoing repair and contributes to PCNA stimulation of DNA ligation. DNA ligase is conformationally flexible within the complex, with its domains fully ordered only when encircling the repaired DNA to form a stacked ring structure with PCNA. The structures highlight DNA ligase structural transitions while docked on PCNA, changes in DNA conformation during ligation, and the potential for DNA ligase domains to regulate PCNA accessibility to other repair factors.
Topics: Cryoelectron Microscopy; DNA; DNA Ligase ATP; DNA Ligases; DNA Replication; Nucleic Acid Conformation; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 34838188
DOI: 10.1016/j.str.2021.11.002 -
Chembiochem : a European Journal of... Nov 2021Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for...
Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3' ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3' overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5' overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.
Topics: Cloning, Molecular; DNA; Deoxyuridine; Gene Library; Oligonucleotides
PubMed: 34547157
DOI: 10.1002/cbic.202100425 -
Nucleic Acids Research May 2022DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To...
DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.
Topics: DNA; DNA Ligases; Humans; Purines
PubMed: 35438779
DOI: 10.1093/nar/gkac241 -
Biosensors & Bioelectronics Aug 2022For the 16S rRNA gene of bacterial analysis, the current usage of single recognition probe always causes the false positive result. Meanwhile, it is usually impossible...
For the 16S rRNA gene of bacterial analysis, the current usage of single recognition probe always causes the false positive result. Meanwhile, it is usually impossible for direct ligation of two free DNA strands modified with click ligation groups in the solution. In our work, A DNA tetrahedron supported click ligation has been elaborately designed; thereby a new method has been further developed for bacterial analysis with dual recognition on two target regions of 16S rRNA gene. Compared with free click ligation, DNA tetrahedron supported click ligation exhibits high reaction rate and ligation efficiency as a result of proximity effect on the supporting interface. The designed DNA tetrahedron can simultaneously bind with two target regions of 16S rRNA gene in bacteria, inducing the proximity of reaction groups and efficient occurrence of click ligation. The established method shows the practical applicability in the serum sample. In a word, inspired by high ligation efficiency on the interface, DNA tetrahedron supported click ligation has been firstly developed and served for bacterial analysis through dual recognition with high specificity, high sensitivity and good performance.
Topics: Bacteria; Biosensing Techniques; Click Chemistry; DNA; RNA, Ribosomal, 16S
PubMed: 35561579
DOI: 10.1016/j.bios.2022.114342 -
Nature May 2021DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone...
DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors. Here we applied single-particle cryo-electron microscopy to visualize two key DNA-protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.
Topics: Cryoelectron Microscopy; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Repair Enzymes; DNA-Activated Protein Kinase; DNA-Binding Proteins; Humans; Ku Autoantigen; Models, Molecular; Phosphorylation
PubMed: 33854234
DOI: 10.1038/s41586-021-03458-7 -
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 -
Genome Research Jul 2023The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can...
The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can access, cut, and ligate adapters to DNA fragments for subsequent amplification and sequencing. These sequenced regions are quantified and tested for enrichment in a process referred to as "peak calling." Most unsupervised peak calling methods are based on simple statistical models and suffer from elevated false positive rates. Newly developed supervised deep learning methods can be successful, but they rely on high quality labeled data for training, which can be difficult to obtain. Moreover, though biological replicates are recognized to be important, there are no established approaches for using replicates in the deep learning tools, and the approaches available for traditional methods either cannot be applied to ATAC-seq, where control samples may be unavailable, or are post hoc and do not capitalize on potentially complex, but reproducible signal in the read enrichment data. Here, we propose a novel peak caller that uses unsupervised contrastive learning to extract shared signals from multiple replicates. Raw coverage data are encoded to obtain low-dimensional embeddings and optimized to minimize a contrastive loss over biological replicates. These embeddings are passed to another contrastive loss for learning and predicting peaks and decoded to denoised data under an autoencoder loss. We compared our replicative contrastive learner (RCL) method with other existing methods on ATAC-seq data, using annotations from ChromHMM genomic labels and transcription factor ChIP-seq as noisy truth. RCL consistently achieved the best performance.
Topics: Chromatin Immunoprecipitation Sequencing; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Chromatin; DNA
PubMed: 37217250
DOI: 10.1101/gr.277677.123 -
Nanoscale Oct 2021Within the field of DNA nanotechnology, numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging...
Within the field of DNA nanotechnology, numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging applications. These structures typically suffer from a low tolerance against non-optimal environmental conditions including elevated temperatures. Here, we apply a chemical ligation method to covalently seal the nicks between adjacent 5' phosphorylated and 3' amine-modified strands within the DNA nanostructures. Using a cost-effective enzymatic strand modification procedure, we are able to batch-modify all DNA strands even of large DNA objects, such as origami nanostructures. The covalent strand linkage increases the temperature stability of the structures by ∼10 K. Generally, our method also allows a 'surgical' introduction of covalent strand linkages at preselected positions. It can also be used to map the strand ligation into chains throughout the whole nanostructure and identify assembly defects. We expect that our method can be applied to a large variety of DNA nanostructures, in particular when full control over the introduced covalent linkages and the absence of side adducts and DNA damages are required.
Topics: DNA; Nanostructures; Nanotechnology; Nucleic Acid Conformation; Temperature
PubMed: 34657945
DOI: 10.1039/d1nr04225d -
Applied Microbiology and Biotechnology Feb 2021It is of great significance to establish sensitive and accurate pathogen detection methods, considering the continuous emergence or re-emergence of infectious diseases... (Review)
Review
It is of great significance to establish sensitive and accurate pathogen detection methods, considering the continuous emergence or re-emergence of infectious diseases seriously influences the safety of human and animals. Proximity ligation assay (PLA) is developed for the sensitive protein detection and also can be used for the detection of pathogens. PLA employs aptamer or monoclonal/polyclonal antibody-nucleic acid complexes as proximity probes. When the paired proximity probes bind to the same target protein or protein complex, they will be adjacent to each other and form an amplifiable DNA sequence through ligation. Combining the specificity of enzyme-linked immunosorbent assay (ELISA) and sensitivity of polymerase chain reaction (PCR), PLA transforms the detection of protein into the detection of DNA nucleic acid sequence. Therefore, as an ultrasensitive protein assay, PLA has great potential for quantification, localization of protein, and clinical diagnostics. In this review, we summarize the basic principles of PLA and its applications in pathogen detection. KEY POINTS: • Different forms of proximity ligation assay are introduced. • Applications of proximity ligation assay in pathogen detection are summarized. • Proximity ligation assay is an ultrasensitive method to quantify protein and pathogen.
Topics: Animals; Biological Assay; DNA; Enzyme-Linked Immunosorbent Assay; Humans; Oligonucleotides; Polymerase Chain Reaction
PubMed: 33427935
DOI: 10.1007/s00253-020-11049-1