-
Plant Physiology Feb 2016Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced...
Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced GT in plants remains challenging due to inefficient delivery of HDR templates and SSNs into plant nuclei. In many plants, including rice, Agrobacterium-mediated transformation is the most practical means of transformation because this biotic transformation system can deliver longer and more intact DNA payloads with less incorporation of fragmented DNA compared with physical transformation systems such as polyethylene glycol, electroporation, or biolistics. Following infection with Agrobacterium, transfer of transfer DNA (T-DNA) to the nucleus and its integration into the plant genome occur consecutively during cocultivation, thus timing the induction of DNA double-strand breaks (DSBs) on the target gene to coincide with the delivery of the HDR template is crucial. To synchronize DSB induction and delivery of the HDR template, we transformed a Cas9 expression construct and GT vector harboring the HDR template with guide RNAs (gRNAs) targeting the rice acetolactate synthase (ALS) gene either separately or sequentially into rice calli. When gRNAs targeting ALS were transcribed transiently from double-stranded T-DNA containing the HDR template, DSBs were induced in the ALS locus by the assembled Cas9/gRNA complex and homologous recombination was stimulated. Contrary to our expectations, there was no great difference in GT efficiency between Cas9-expressing and nonexpressing cells. However, when gRNA targeting DNA ligase 4 was transformed with Cas9 prior to the GT experiment, GT efficiency increased dramatically and more than one line exhibiting biallelic GT at the ALS locus was obtained.
Topics: Acetolactate Synthase; Agrobacterium; CRISPR-Cas Systems; DNA Breaks, Double-Stranded; DNA Ligases; DNA, Bacterial; Gene Targeting; Genome, Plant; Homologous Recombination; Oryza; Plant Proteins; Transformation, Genetic
PubMed: 26668334
DOI: 10.1104/pp.15.01663 -
DNA Repair Oct 2021The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to...
The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligase activities.
Topics: Animals; Cell Line; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligases; DNA-Activated Protein Kinase; HeLa Cells; Humans; Nucleosomes; Spodoptera; Xenopus
PubMed: 34339948
DOI: 10.1016/j.dnarep.2021.103193 -
Critical Reviews in Biotechnology Sep 2020Biosensor devices are important in clinical practice and environmental studies because they allow specific detection of target molecules from a variety of samples.... (Review)
Review
Biosensor devices are important in clinical practice and environmental studies because they allow specific detection of target molecules from a variety of samples. However, challenges still remain when attempting to develop sensitive, selective, rapid, and cost-effective assays for biomolecule detection. Devices that use DNA-modifying enzymes to catalyze detection reactions have recently been developed. These devices show promise because they are often more sensitive and specific, have shorter assay times, are more cost-effective, and are easier to use than the currently used biosensors. Here, we review the current trends in DNA-modifying enzyme reaction-coupled biosensors, including devices using DNA polymerases, nicking endonuclease, exonucleases, and ligases. The molecular strategies underlying diverse DNA-modifying enzymes coupled to biomolecule detection platforms are reviewed. We also discuss the strengths and limitations of each strategy and suggest methods to overcome current limitations. Finally, the future prospects of DNA-modifying enzyme reaction-coupled biosensor development have been proposed.
Topics: Biosensing Techniques; DNA Ligases; DNA-Directed DNA Polymerase; Humans; Molecular Diagnostic Techniques
PubMed: 32429779
DOI: 10.1080/07388551.2020.1764485 -
The Journal of Biological Chemistry Aug 2021Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining...
Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus.
Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.
Topics: Catalytic Domain; DNA Damage; DNA Ligase ATP; DNA Repair; Humans; Phosphorylation; Poly-ADP-Ribose Binding Proteins
PubMed: 34181949
DOI: 10.1016/j.jbc.2021.100921 -
Protein Science : a Publication of the... Sep 2021Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic... (Review)
Review
Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non-homologous end joining (NHEJ) as the primary conserved DNA double-strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB-inducing agents, generation of antibody and T-cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X-ray scattering (SAXS) results combined with X-ray crystallography (MX) and cryo-electron microscopy (cryo-EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra-molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo-EM or MX structures. In the long-range synaptic complex, X-ray repair cross-complementing 4 (XRCC4) plus XRCC4-like-factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto-phosphorylation opens DNA-PKcs dimer licensing NHEJ via concerted conformational transformations of XLF-XRCC4, XLF-Ku80, and LigIV -Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short-range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation-of-function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.
Topics: Binding Sites; DNA Breaks, Double-Stranded; DNA Ligase ATP; DNA Repair Enzymes; DNA, Neoplasm; DNA-Activated Protein Kinase; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; Kinetics; Ku Autoantigen; Models, Molecular; Neoplasms; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Substrate Specificity
PubMed: 34056803
DOI: 10.1002/pro.4133 -
Current Medicinal Chemistry 2018The clustered DNA lesions are a characteristic feature of ionizing radiation and are defined as two or more damage sites formed within 20 bps after the passage of a... (Review)
Review
The clustered DNA lesions are a characteristic feature of ionizing radiation and are defined as two or more damage sites formed within 20 bps after the passage of a single radiation track. The clustered DNA lesions are divided into two major groups: double-stranded breaks (DSBs) and non-DSB clusters also known as Oxidatively-induced Clustered DNA Lesions (OCDLs), which could involve either two opposing strands or the same strand. As irradiation is gaining greater interest in cancer treatment as well as in imaging techniques, the detailed knowledge of its genotoxicity and the mechanisms of repair of radiation-induced DNA damage remain issues to explore. In this review we look at the ways the cell copes with clustered DNA lesions, especially with 5',8-cyclo-2'-deoxypurines. As the base excision repair deals with isolated lesions, complex damage is more difficult to repair. Depending on the number of lesions within a cluster, their types and mutual distribution, long-patch BER or NER are activated. During the repair of opposing lesions, DSBs could be generated, which are repaired either by nonhomologous end joining (NHEJ) or homologous recombination (HR). The repair of individual lesions within a cluster progresses gradually. This slower processing of particular damage might lead to severe biological consequences such as misrepair, mutations and chromosomal rearrengement as it enhances the plausibility of a cluster encountering a replication fork prior to its repair. The consequences of clustered DNA lesions on cell survival and their relevance to the efficacy and safety of radiotherapy and radiodiagnosis will also be discussed.
Topics: DNA; DNA Adducts; DNA Damage; DNA Glycosylases; DNA Ligase ATP; DNA Repair; Humans; Neoplasms; Poly-ADP-Ribose Binding Proteins; Radiation, Ionizing
PubMed: 29484975
DOI: 10.2174/0929867325666180226110502 -
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 -
DNA Repair Nov 2014In mammals, NAD represents a nodal point for metabolic regulation, and its availability is critical to genome stability. Several NAD-consuming enzymes are induced in... (Review)
Review
In mammals, NAD represents a nodal point for metabolic regulation, and its availability is critical to genome stability. Several NAD-consuming enzymes are induced in various stress conditions and the consequent NAD decline is generally accompanied by the activation of NAD biosynthetic pathways to guarantee NAD homeostasis. In the bacterial world a similar scenario has only recently begun to surface. Here we review the current knowledge on the involvement of NAD homeostasis in bacterial stress response mechanisms. In particular, we focus on the participation of both NAD-consuming enzymes (DNA ligase, mono(ADP-ribosyl) transferase, sirtuins, and RNA 2'-phosphotransferase) and NAD biosynthetic enzymes (both de novo, and recycling enzymes) in the response to DNA/RNA damage. As further supporting evidence for such a link, a genomic context analysis is presented showing several conserved associations between NAD homeostasis and stress responsive genes.
Topics: ADP Ribose Transferases; Adenosine Diphosphate Ribose; Bacteria; DNA Damage; DNA Ligases; DNA, Bacterial; Group III Histone Deacetylases; Homeostasis; NAD; Niacinamide; RNA, Bacterial
PubMed: 25127744
DOI: 10.1016/j.dnarep.2014.07.014 -
Organic & Biomolecular Chemistry Feb 2019Ligase-catalyzed oligonucleotide polymerisations (LOOPER) can readily generate libraries of diversely-modified nucleic acid polymers, which can be subjected to iterative...
Ligase-catalyzed oligonucleotide polymerisations (LOOPER) can readily generate libraries of diversely-modified nucleic acid polymers, which can be subjected to iterative rounds of in vitro selection to evolve functional activity. While there exist several different DNA ligases, T4 DNA ligase has most often been used for the process. Recently, T3 DNA ligase was shown to be effective in LOOPER; however, little is known about the fidelity and efficiency of this enzyme in LOOPER. In this paper we evaluate the efficiency of T3 DNA ligase and T4 DNA ligase for various codon lengths and compositions within the context of polymerisation fidelity and yield. We find that T3 DNA ligase exhibits high efficiency and fidelity with short codon lengths, but struggles with longer and more complex codon libraries, while T4 DNA ligase exhibits the opposite trend. Interestingly, T3 DNA ligase is unable to accommodate modifications at the 8-position of adenosine when integrated into short codons, which will create challenges in expanding the available codon set for the process. The limitations and strengths of the two ligases are further discussed within the context of LOOPER.
Topics: Biocatalysis; DNA Ligases; Oligonucleotides; Polymerization
PubMed: 30357247
DOI: 10.1039/c8ob01958d -
European Journal of Medicinal Chemistry Nov 2019The emergence of drug resistance, coupled with the issue of low tumor selectivity and toxicity is a major pitfall in cancer chemotherapy. It has necessitated the urgent... (Review)
Review
The emergence of drug resistance, coupled with the issue of low tumor selectivity and toxicity is a major pitfall in cancer chemotherapy. It has necessitated the urgent need for the discovery of less toxic and more potent new anti-cancer pharmaceuticals, which target the interactive mechanisms involved in division and metastasis of cancer cells. Human DNA ligase I (hligI) plays an important role in DNA replication by linking Okazaki fragments on the lagging strand of DNA, and also participates in DNA damage repair processes. Dysregulation of the functioning of such ligases can severely impact DNA replication and repair pathways events that are generally targeted in cancer treatment. Although, several human DNA ligase inhibitors have been reported in the literature but unfortunately not a single inhibitor is currently being used in cancer chemotherapy. Results of pre-clinical studies also support the fact that human DNA ligases are an attractive target for the development of new anticancer agents which work by the selective inhibition of rapidly proliferating cancer cells. In this manuscript, we discuss, in brief, the structure, synthesis, structure-activity-relationship (SAR) and anticancer activity of recently reported hLigI inhibitors.
Topics: Antineoplastic Agents; Cell Proliferation; DNA Ligase ATP; Enzyme Inhibitors; Humans; Neoplasms; Structure-Activity Relationship
PubMed: 31499361
DOI: 10.1016/j.ejmech.2019.111657