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Animal Cells and Systems 2023Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs)...
Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.
PubMed: 37808549
DOI: 10.1080/19768354.2023.2265165 -
Journal of Molecular Cell Biology Feb 2021Ever since gene targeting or specific modification of genome sequences in mice was achieved in the early 1980s, the reverse genetic approach of precise editing of any... (Review)
Review
Ever since gene targeting or specific modification of genome sequences in mice was achieved in the early 1980s, the reverse genetic approach of precise editing of any genomic locus has greatly accelerated biomedical research and biotechnology development. In particular, the recent development of the CRISPR/Cas9 system has greatly expedited genetic dissection of 3D genomes. CRISPR gene-editing outcomes result from targeted genome cleavage by ectopic bacterial Cas9 nuclease followed by presumed random ligations via the host double-strand break repair machineries. Recent studies revealed, however, that the CRISPR genome-editing system is precise and predictable because of cohesive Cas9 cleavage of targeting DNA. Here, we synthesize the current understanding of CRISPR DNA fragment-editing mechanisms and recent progress in predictable outcomes from precise genetic engineering of 3D genomes. Specifically, we first briefly describe historical genetic studies leading to CRISPR and 3D genome engineering. We then summarize different types of chromosomal rearrangements by DNA fragment editing. Finally, we review significant progress from precise 1D gene editing toward predictable 3D genome engineering and synthetic biology. The exciting and rapid advances in this emerging field provide new opportunities and challenges to understand or digest 3D genomes.
Topics: Animals; CRISPR-Cas Systems; DNA; Gene Editing; Genetic Engineering; Genome; Humans; Imaging, Three-Dimensional
PubMed: 33125070
DOI: 10.1093/jmcb/mjaa060 -
Journal of Visualized Experiments : JoVE Jan 2023Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix...
Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population. A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.
Topics: Chromosomes; Chromatin; DNA; Cell Nucleus; DNA Restriction Enzymes; Formaldehyde; Nucleic Acid Conformation
PubMed: 36744801
DOI: 10.3791/64001 -
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 -
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 -
Biomolecules Mar 2022Ubiquitination is controlled by a series of E1, E2, and E3 enzymes that can ligate ubiquitin to cellular proteins and dictate the turnover of a substrate and the outcome... (Review)
Review
Ubiquitination is controlled by a series of E1, E2, and E3 enzymes that can ligate ubiquitin to cellular proteins and dictate the turnover of a substrate and the outcome of signalling events such as DNA damage repair and cell cycle. This process is complex due to the combinatorial power of ~35 E2 and ~1000 E3 enzymes involved and the multiple lysine residues on ubiquitin that can be used to assemble polyubiquitin chains. Recently, mass spectrometric methods have identified that most enzymes in the ubiquitination cascade can be further modified through acetylation or phosphorylation under particular cellular conditions and altered modifications have been noted in different cancers and neurodegenerative diseases. This review provides a cohesive summary of ubiquitination, acetylation, and phosphorylation sites in ubiquitin, the human E1 enzyme UBA1, all E2 enzymes, and some representative E3 enzymes. The potential impacts these post-translational modifications might have on each protein function are highlighted, as well as the observations from human disease.
Topics: Acetylation; Humans; Phosphorylation; Polyubiquitin; Protein Processing, Post-Translational; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 35327659
DOI: 10.3390/biom12030467 -
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 -
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 -
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 -
Genes Oct 2019DNA topoisomerase II (TOP2) plays a critical role in many processes such as replication and transcription, where it resolves DNA structures and relieves torsional... (Review)
Review
DNA topoisomerase II (TOP2) plays a critical role in many processes such as replication and transcription, where it resolves DNA structures and relieves torsional stress. Recent evidence demonstrated the association of TOP2 with topologically associated domains (TAD) boundaries and CCCTC-binding factor (CTCF) binding sites. At these sites, TOP2 promotes interactions between enhancers and gene promoters, and relieves torsional stress that accumulates at these physical barriers. Interestingly, in executing its enzymatic function, TOP2 contributes to DNA fragility through re-ligation failure, which results in persistent DNA breaks when unrepaired or illegitimately repaired. Here, we discuss the biological processes for which TOP2 is required and the steps at which it can introduce DNA breaks. We describe the repair processes that follow removal of TOP2 adducts and the resultant broken DNA ends, and present how these processes can contribute to disease-associated mutations. Furthermore, we examine the involvement of TOP2-induced breaks in the formation of oncogenic translocations of leukemia and papillary thyroid cancer, as well as the role of TOP2 and proteins which repair TOP2 adducts in other diseases. The participation of TOP2 in generating persistent DNA breaks and leading to diseases such as cancer, could have an impact on disease treatment and prevention.
Topics: CCCTC-Binding Factor; Chromatin; DNA; DNA Breaks, Double-Stranded; DNA Repair; DNA Topoisomerases, Type II; Humans; Leukemia, Myeloid, Acute; Thyroid Cancer, Papillary; Topoisomerase II Inhibitors; Torsion, Mechanical
PubMed: 31614754
DOI: 10.3390/genes10100791