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Virology Jul 1996Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with...
Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.
Topics: Adenosine Triphosphate; DNA; DNA Ligases; DNA, Single-Stranded; Escherichia coli; Kinetics; Oligodeoxyribonucleotides; Recombinant Proteins; Vaccinia virus
PubMed: 8661420
DOI: 10.1006/viro.1996.0358 -
Bioorganic & Medicinal Chemistry Jun 2019Quinone methides (QMs) are transient reactive species that can be efficiently generated from stable precursors under a variety of biocompatible conditions. Due to their... (Review)
Review
Quinone methides (QMs) are transient reactive species that can be efficiently generated from stable precursors under a variety of biocompatible conditions. Due to their electrophilic nature, QMs have been widely explored as cross-linking agents of DNA and proteins under physiological conditions. However, QMs also have a diene character and can irreversibly react via Diels-Alder reaction with electron-rich dienophiles. This particular reactivity has been recently exploited to label biomolecules with fluorophores in living cells. QMs are characterised by two unique properties that make them ideal candidates for chemical biology applications: i) they can be efficiently generated in situ from very stable precursors by means of bio-orthogonal protocols ii) they are reversible cross-linking agents, making them suitable for "catch and release" target-enrichment experiments. Nevertheless, there are only few examples reported to date that truly take advantage of QMs unique chemistry in the context of chemical-biology assay development. In this review, we will examine the most relevant examples that illustrate the benefit of using QMs for chemical biology purposes and we will anticipate novel approaches to further their applications in biologically relevant contexts.
Topics: Alkylation; Click Chemistry; Cycloaddition Reaction; DNA; Humans; Indolequinones; Microscopy, Fluorescence; Proteins; RNA Interference; Ultraviolet Rays
PubMed: 30955994
DOI: 10.1016/j.bmc.2019.04.001 -
Angewandte Chemie (International Ed. in... Dec 2021DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are...
DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site-specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3-DNA crosslinks blocked DNA synthesis by hPol η polymerase, but were bypassed following proteolytic processing.
Topics: Cytosine; DNA; DNA-Directed DNA Polymerase; Histones; Humans; Molecular Structure
PubMed: 34634172
DOI: 10.1002/anie.202109418 -
Accounts of Chemical Research Dec 2020Ribozymes and deoxyribozymes are catalytic RNA and DNA, respectively, that catalyze chemical reactions such as self-cleavage or ligation reactions. While some ribozymes...
Ribozymes and deoxyribozymes are catalytic RNA and DNA, respectively, that catalyze chemical reactions such as self-cleavage or ligation reactions. While some ribozymes are found in nature, a larger variety of ribozymes and deoxyribozymes have been discovered by in vitro selection from random sequences. These catalytic nucleic acids, especially ribozymes, are of fundamental interest because they are crucial for the RNA world hypothesis, which suggests that RNA played a central role in both the propagation of genetic information and catalyzing metabolic reactions in primordial life prior to the emergence of proteins and DNA. On the practical side, catalytic nucleic acids have been extensively engineered for various applications, such as biosensors and genetic devices for synthetic biology. Therefore, it is important to gain a deeper understanding of the sequence-function relationships of ribozymes and deoxyribozymes.Mutational analysis, or measurements of activities of catalytic nucleic acid mutants, is one of the most fundamental approaches for that purpose. Mutations that abolish, reduce, retain, or even increase activity provide useful information about nucleic acid catalysts for engineering and other purposes. However, methods for mutational analysis of ribozymes and deoxyribozymes have not evolved much for decades, requiring tedious and low-throughput assays (e.g., gel electrophoresis) of individually prepared mutants. This has prevented researchers from performing quantitative mutational analysis of ribozymes and deoxyribozymes on a large scale.To address this limitation, we developed a massively parallel ribozyme and deoxyribozyme assay strategy that allows >10 assays using high-throughput sequencing (HTS). We used HTS to literally count the number of cleaved (or ligated) and uncleaved (or unligated) ribozyme (or deoxyribozyme) sequences and calculated the activities of each mutant in a reaction mixture. This simple yet powerful strategy was applied to analyze the mutational effects of various natural and synthetic ribozymes and deoxyribozymes at scales impossible for conventional mutational analysis. These large-scale sequence-function data sets were used to better understand the functional consequences of mutations and to engineer ribozymes for practical applications. Furthermore, these newly available data are motivating researchers to employ more rigorous computational methods to extract additional insights such as structural information and nonlinear effects of multiple mutations. The new HTS-based assay strategy is distinct from and complementary to a related strategy that uses HTS to analyze ribozyme and deoxyribozyme populations subjected to in vitro selection. Postselection sequencing can cover a larger sequence space, although it does not directly quantify the activities of ribozyme and deoxyribozyme mutants. With further advances in DNA sequencing technologies and computational methods, there should be more opportunities to harness the power of HTS to deepen our understanding of catalytic nucleic acids and enhance our ability to engineer them for even more applications.
Topics: Aptamers, Nucleotide; DNA, Catalytic; High-Throughput Nucleotide Sequencing; High-Throughput Screening Assays; Kinetics; Mutation; Nucleic Acid Conformation; RNA, Catalytic
PubMed: 33164502
DOI: 10.1021/acs.accounts.0c00546 -
Nature Communications Feb 2021Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of...
Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chemical ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also observed. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.
Topics: Amino Alcohols; Base Pairing; Biocatalysis; Butylene Glycols; Cations, Divalent; DNA; DNA Primers; Imidazoles; Manganese; Nucleic Acid Conformation; Nucleic Acids; RNA; Solutions
PubMed: 33547322
DOI: 10.1038/s41467-021-21128-0 -
ACS Synthetic Biology Oct 2023Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single- or...
Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single- or double-stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product recircularization using restriction and ligation, leading to a higher amplification yield and enabling the generation of circular products. However, C2CA is a multistep, nonisothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37 °C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.
Topics: DNA; DNA, Circular; Nucleic Acid Amplification Techniques
PubMed: 37729629
DOI: 10.1021/acssynbio.3c00390 -
Methods in Molecular Biology (Clifton,... 2019We recently developed a method for assessing RNA-DNA interactions using proximity ligation assays (PLA). This technique, termed the "RNA-DNA interaction assay" (RDIA),...
We recently developed a method for assessing RNA-DNA interactions using proximity ligation assays (PLA). This technique, termed the "RNA-DNA interaction assay" (RDIA), involves differentially labeling DNA and RNA with EdU and BrU, respectively. Once labeled, PLA is performed to assess if the labeled molecules are in close proximity. Here we provide a detailed description of the modified RDIA protocol utilizing currently commercially available BrdU antibodies. As an example, we show its ability to detect nascent transcripts on recently synthesized DNA in both cultured H1299 cells and mouse embryonic stem cells.
Topics: Animals; Antibodies; Bromodeoxyuridine; Cell Line; DNA; Humans; Mice; Mouse Embryonic Stem Cells; RNA
PubMed: 31124093
DOI: 10.1007/978-1-4939-9537-0_10 -
Current Biology : CB Jun 2021Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of...
Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a collapsed replication fork comprising a single-ended double-strand break and a 'hybrid nick' with parental DNA on one side and nascent DNA on the other (Figure 1A). We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a fork from an adjacent origin can promote localized re-replication if the hybrid nick is ligated. We have modelled this situation with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA Ligases; DNA Replication; DNA-Directed DNA Polymerase; Escherichia coli; Genome; Multienzyme Complexes; Replication Origin; Yeasts
PubMed: 34102115
DOI: 10.1016/j.cub.2021.03.043 -
Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.American Journal of Clinical Pathology Jan 2017Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent...
OBJECTIVES
Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods.
METHODS
Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared.
RESULTS
Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance.
CONCLUSIONS
FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.
Topics: DNA; Formaldehyde; Humans; Multiplex Polymerase Chain Reaction; Paraffin Embedding; Tissue Fixation
PubMed: 28122725
DOI: 10.1093/ajcp/aqw205 -
BioTechniques Jan 2007DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of...
DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of subsequent manipulations (e.g., selection via bacterial transformation) often obviates the need for a high yield of correctly ligated products. However the ability to perform high-yield, preparative-scale DNA ligations would benefit a number of downstream applications ranging from standard molecular cloning to biophysics and DNA computing. We describe here a ligation technique that specifically converts off-pathway ligation products back into substrate. We term this second-chance strategy enzymatic ligation assisted by nucleases (ELAN) and demonstrate the ordered assembly of four DNA fragments via simultaneous ligation and digestion in the presence of eight restriction enzymes. Use of ELAN increased the yield of the desired product by more than 30-fold.
Topics: DNA; DNA Restriction Enzymes; Deoxyribonucleases; Genetic Techniques
PubMed: 17269489
DOI: 10.2144/000112283