-
Nature Communications Nov 2019DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1...
DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3'-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg-reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg, and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage.
Topics: DNA; DNA Breaks, Single-Stranded; DNA Damage; DNA Ligase ATP; DNA Repair; DNA Replication; DNA-Binding Proteins; Guanine; Humans; Magnesium; Nuclear Proteins; Nucleic Acid Conformation; Oxidation-Reduction; Protein Structure, Tertiary; Recombinational DNA Repair
PubMed: 31780661
DOI: 10.1038/s41467-019-13478-7 -
F1000Research 2019The relationship between varicoceles and subfertility is well-established, but recent evidence suggests that varicoceles may cause global testicular dysfunction. This... (Review)
Review
The relationship between varicoceles and subfertility is well-established, but recent evidence suggests that varicoceles may cause global testicular dysfunction. This has led to exploration into expanding the indications for varicocelectomy. This review examines the literature regarding varix ligation as a treatment for non-obstructive azoospermia, elevated DNA fragmentation, and hypogonadism.
Topics: Azoospermia; DNA Fragmentation; Humans; Hypogonadism; Ligation; Male; Testis; Varicocele
PubMed: 31543949
DOI: 10.12688/f1000research.19579.1 -
Journal of Molecular Biology May 2019DNA ligases are a highly conserved group of nucleic acid enzymes that play an essential role in DNA repair, replication, and recombination. This review focuses on... (Review)
Review
DNA ligases are a highly conserved group of nucleic acid enzymes that play an essential role in DNA repair, replication, and recombination. This review focuses on functional interaction between DNA polymerases and DNA ligases in the repair of single- and double-strand DNA breaks, and discusses the notion that the substrate channeling during DNA polymerase-mediated nucleotide insertion coupled to DNA ligation could be a mechanism to minimize the release of potentially mutagenic repair intermediates. Evidence suggesting that DNA ligases are essential for cell viability includes the fact that defects or insufficiency in DNA ligase are casually linked to genome instability. In the future, it may be possible to develop small molecule inhibitors of mammalian DNA ligases and/or their functional protein partners that potentiate the effects of chemotherapeutic compounds and improve cancer treatment outcomes.
Topics: Animals; DNA; DNA Breaks; DNA Ligases; DNA Repair; DNA-Directed DNA Polymerase; Genomic Instability; Humans; Protein Interaction Maps
PubMed: 31034893
DOI: 10.1016/j.jmb.2019.04.028 -
Journal of Visualized Experiments : JoVE Jun 2022The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5...
The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.
Topics: Chromatin; Chromatin Immunoprecipitation Sequencing; DNA; Epigenesis, Genetic; High-Throughput Nucleotide Sequencing; Neoplasms; Sequence Analysis, DNA; Transcription Factors
PubMed: 35848835
DOI: 10.3791/64242 -
Environmental and Molecular Mutagenesis Jan 2015Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the... (Review)
Review
Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini.
Topics: Animals; DNA; DNA Damage; DNA Repair; Disease Progression; Genomic Instability; Humans; Nucleic Acid Conformation
PubMed: 25111769
DOI: 10.1002/em.21892 -
The Journal of Biological Chemistry Jul 2023Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein...
Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380 bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70 ± 3 h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.
Topics: Nucleosomes; DNA; Mechanical Phenomena; Biophysical Phenomena; Polymerase Chain Reaction
PubMed: 37257819
DOI: 10.1016/j.jbc.2023.104874 -
Journal of the American Chemical Society Aug 2023Previously, nonenzymatic primer extension reaction of l-threoninol nucleic acid (L-TNA) was achieved in the presence of -cyanoimidazole (CNIm) and Mn; however, the...
Previously, nonenzymatic primer extension reaction of l-threoninol nucleic acid (L-TNA) was achieved in the presence of -cyanoimidazole (CNIm) and Mn; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-TNA to L-TNA and of DNA to DNA. We found that Cd, Ni, and Co accelerated ligation of both L-TNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-TNA fragment and template, resulting in unexpectedly more effective L-TNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-TNA primer could be elongated by ligation to L-TNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.
Topics: Nucleic Acids; DNA; Amino Alcohols; RNA; Nucleic Acid Conformation
PubMed: 37466125
DOI: 10.1021/jacs.3c04979 -
DNA Repair Oct 2023DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the... (Review)
Review
DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the majority of these breaks in vertebrates by directly ligating DNA ends back together. Upon formation of a DSB, a multiprotein complex is assembled on DNA ends which tethers them together within a synaptic complex. Synapsis is a critical step of the NHEJ pathway as loss of synapsis can result in mispairing of DNA ends and chromosome translocations. As DNA ends are commonly incompatible for ligation, the NHEJ machinery must also process ends to enable rejoining. This review describes how recent progress in single-molecule approaches and cryo-EM have advanced our molecular understanding of DNA end synapsis during NHEJ and how synapsis is coordinated with end processing to determine the fidelity of repair.
Topics: Animals; DNA End-Joining Repair; DNA; DNA-Binding Proteins; DNA Breaks, Double-Stranded; Chromosome Pairing; DNA Repair
PubMed: 37572577
DOI: 10.1016/j.dnarep.2023.103553 -
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 -
Journal of Molecular Biology Dec 2020More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA...
More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5' end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.
Topics: DNA; DNA Ligase ATP; DNA Ligases; DNA Polymerase I; DNA Polymerase III; DNA Replication; Flap Endonucleases; Genome, Human; Holoenzymes; Humans; Multiprotein Complexes; Proliferating Cell Nuclear Antigen; Protein Binding; Replication Protein C
PubMed: 33157085
DOI: 10.1016/j.jmb.2020.10.032