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PLoS Biology Mar 2021Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a...
Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.
Topics: 3' Untranslated Regions; Animals; DNA; DNA Breaks, Double-Stranded; DNA Repair; DNA Replication; DNA-Binding Proteins; Genome; Humans; Sequence Analysis, DNA
PubMed: 33760805
DOI: 10.1371/journal.pbio.3000886 -
Methods in Molecular Biology (Clifton,... 2022DEL technology is dependent on the generation and analysis of large amounts of DNA sequence data. In this chapter, we describe a method to sequence DEL libraries that...
DEL technology is dependent on the generation and analysis of large amounts of DNA sequence data. In this chapter, we describe a method to sequence DEL libraries that uses a customized preparative PCR protocol, along with standard steps for purification, analysis, and sequencing of the amplified library DNA on an Illumina sequencing platform. Compared with standard Illumina sequencing library preparation protocols, in which a PCR reaction is followed by end repair, adenylation, ligation of Illumina adapters, and a second preparative PCR reaction, the customized operations described here provide significantly improved process efficiency and sequencing quality.
Topics: DNA; Gene Library; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 36083556
DOI: 10.1007/978-1-0716-2545-3_21 -
Proceedings of the National Academy of... Dec 2022The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The...
The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.
Topics: Poly(ADP-ribose) Polymerase Inhibitors; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Homologous Recombination; DNA
PubMed: 36442094
DOI: 10.1073/pnas.2214935119 -
Critical Reviews in Analytical Chemistry 2023DNA biosensors play important roles in environmental, medical, industrial and agricultural analysis. Many DNA biosensors have been designed based on the enzyme catalytic... (Review)
Review
DNA biosensors play important roles in environmental, medical, industrial and agricultural analysis. Many DNA biosensors have been designed based on the enzyme catalytic reaction. Because of the importance of enzymes in biosensors, we present a review on this topic. In this review, the enzymes were divided into DNAzymes and nucleases according to their chemical nature. Firstly, we introduced the DNAzymes with different function inducing cleavage, metalation, peroxidase, ligation and allosterism. In this section, the G-quadruplex DNAzyme, as a hot topic in recent years, was described in detail. Then, the nucleases-assisted signal amplification method was also reviewed in three categories including exonucleases, endonucleases and other nucleases according to the digestion sites in DNA substrates. In exonucleases section, the Exo I and Exo III were selected as examples. Then, the DNase I, BamH I, nicking endonuclease, S1 nuclease, the duplex specific nuclease (DSN) and RNases were chosen to illustrate the application of endonucleases. In other nucleases section, DNA polymerases and DNA ligases were detailed. Last, the challenges and future perspectives in the field were discussed.
Topics: DNA, Catalytic; DNA; Biosensing Techniques; Endonucleases
PubMed: 34225516
DOI: 10.1080/10408347.2021.1944046 -
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 -
Mutation Research. Genetic Toxicology... Jan 2022DNA double strand breaks (DSBs) are the most threatening type of DNA lesions and must be repaired properly in order to inhibit severe diseases and cell death. There are... (Review)
Review
DNA double strand breaks (DSBs) are the most threatening type of DNA lesions and must be repaired properly in order to inhibit severe diseases and cell death. There are four major repair pathways for DSBs: non-homologous end joining (NHEJ), homologous recombination (HR), single strand annealing (SSA) and alternative end joining (alt-EJ). Cells choose repair pathway depending on the cell cycle phase and the length of 3' end of the DNA when DSBs are generated. Blunt and short regions of the 5' or 3' overhang DNA are repaired by NHEJ, which uses direct ligation or limited resection processing of the broken DNA end. In contrast, HR, SSA and alt-EJ use the resected DNA generated by the MRN (MRE11-RAD50-NBS1) complex and C-terminal binding protein interacting protein (CtIP) activated during the S and G2 phases. Here, we review recent findings on each repair pathway and the choice of repair mechanism and highlight the role of mismatch repair (MMR) protein in HR.
Topics: DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; G2 Phase; S Phase
PubMed: 35094810
DOI: 10.1016/j.mrgentox.2021.503438 -
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 -
Journal of Molecular Biology Jan 2024The joining of breaks in the DNA phosphodiester backbone is essential for genome integrity. Breaks are generated during normal processes such as DNA replication,... (Review)
Review
The joining of breaks in the DNA phosphodiester backbone is essential for genome integrity. Breaks are generated during normal processes such as DNA replication, cytosine demethylation during differentiation, gene rearrangement in the immune system and germ cell development. In addition, they are generated either directly by a DNA damaging agent or indirectly due to damage excision during repair. Breaks are joined by a DNA ligase that catalyzes phosphodiester bond formation at DNA nicks with 3' hydroxyl and 5' phosphate termini. Three human genes encode ATP-dependent DNA ligases. These enzymes have a conserved catalytic core consisting of three subdomains that encircle nicked duplex DNA during ligation. The DNA ligases are targeted to different nuclear DNA transactions by specific protein-protein interactions. Both DNA ligase IIIα and DNA ligase IV form stable complexes with DNA repair proteins, XRCC1 and XRCC4, respectively. There is functional redundancy between DNA ligase I and DNA ligase IIIα in DNA replication, excision repair and single-strand break repair. Although DNA ligase IV is a core component of the major double-strand break repair pathway, non-homologous end joining, the other enzymes participate in minor, alternative double-strand break repair pathways. In contrast to the nucleus, only DNA ligase IIIα is present in mitochondria and is essential for maintaining the mitochondrial genome. Human immunodeficiency syndromes caused by mutations in either LIG1 or LIG4 have been described. Preclinical studies with DNA ligase inhibitors have identified potentially targetable abnormalities in cancer cells and evidence that DNA ligases are potential targets for cancer therapy.
Topics: Animals; Humans; DNA; DNA Damage; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA Replication; X-ray Repair Cross Complementing Protein 1
PubMed: 37714297
DOI: 10.1016/j.jmb.2023.168276 -
Talanta Mar 2023Proximity ligation assay (PLA) is a vigorously developed homogeneous immunoassay assisted by DNA combining dual recognition of target protein by pairs of proximity... (Review)
Review
Proximity ligation assay (PLA) is a vigorously developed homogeneous immunoassay assisted by DNA combining dual recognition of target protein by pairs of proximity probes, in which the detection of protein is tactfully converted to the detection of DNA. The booming developments in PLA have enabled a variety of ultrasensitive assays for the detection of protein and this concept of PLA is also extended to the detection of nucleic acids and some small molecule. The association between PLA and electrochemical method, defined as electrochemical proximity ligation assay (ECPLA), has gained much interests in disease diagnosis, food safety and environmental assays with the advantages, such as broad range of targets, simplicity, low cost and rapid response. In this review, we took a different perspective to present the history of PLA, the classical ECPLA biosensing methodology as well as the developments of ECPLA based on several key parameters, such as sensitivity, selectivity, reusability and generalization. In addition, the developments of PLA with electrochemiluminescence as readout are also presented. Finally, perspective and some unresolved challenges in ECPLA that can potentially be addressed have also been discussed.
Topics: Proteins; DNA; Immunoassay; Nucleic Acids; Electrochemical Techniques; Biosensing Techniques
PubMed: 36502611
DOI: 10.1016/j.talanta.2022.124158 -
Chemistry (Weinheim An Der Bergstrasse,... Apr 2022DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the...
DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the topological features of nucleosomal DNA and to probe DNA-protein interactions such as HIV-1 and PFV integrases, and DNA gyrase. Here, we synthesized the topologically-interlocked minicircle rotaxane and catenane inside a frame-shaped DNA origami. These minicircles are 183 bp in length, constitute six individual single-stranded DNAs that are ligated to realize duplex interlocking, and adopt temporary base pairing of single strands for interlocking. To probe the DNA-protein interactions, restriction reactions were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically-interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA-protein interactions.
Topics: DNA; DNA Replication; DNA, Circular; DNA, Kinetoplast
PubMed: 35218108
DOI: 10.1002/chem.202200108