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Journal of Molecular Evolution Apr 2022TOPIIA topoisomerases are required for the regulation of DNA topology by DNA cleavage and re-ligation and are important targets of antibiotic and anticancer agents....
TOPIIA topoisomerases are required for the regulation of DNA topology by DNA cleavage and re-ligation and are important targets of antibiotic and anticancer agents. Humans possess two TOPIIA paralogue genes (TOP2A and TOP2B) with high sequence and structural similarity but distinct cellular functions. Despite their functional and clinical relevance, the evolutionary history of TOPIIA is still poorly understood. Here we show that TOPIIA is highly conserved in Metazoa. We also found that TOPIIA paralogues from jawed and jawless vertebrates had different origins related with tetraploidization events. After duplication, TOP2B evolved under a stronger purifying selection than TOP2A, perhaps promoted by the more specialized role of TOP2B in postmitotic cells. We also detected genetic signatures of positive selection in the highly variable C-terminal domain (CTD), possibly associated with adaptation to cellular interactions. By comparing TOPIIA from modern and archaic humans, we found two amino acid substitutions in the TOP2A CTD, suggesting that TOP2A may have contributed to the evolution of present-day humans, as proposed for other cell cycle-related genes. Finally, we identified six residues conferring resistance to chemotherapy differing between TOP2A and TOP2B. These six residues could be targets for the development of TOP2A-specific inhibitors that would avoid the side effects caused by inhibiting TOP2B. Altogether, our findings clarify the origin, diversification and selection pressures governing the evolution of animal TOPIIA.
Topics: Animals; Antigens, Neoplasm; DNA; DNA-Binding Proteins
PubMed: 35165762
DOI: 10.1007/s00239-022-10048-2 -
DNA Repair Oct 2022Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many... (Review)
Review
Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many approaches to gene editing. Ligation of the DSBs by end joining (EJ) pathways can restore the broken chromosome, but the repair junctions can have insertion/deletion (indel) mutations. The indel patterns resulting from DSB EJ are likely defined by the initial structure of the DNA ends, how the ends are processed and synapsed prior to ligation, and the factors that mediate the ligation step. In this review, we describe key factors that influence these steps of DSB EJ in mammalian cells, which is significant both for understanding mutagenesis resulting from clastogenic cancer therapeutics, and for developing approaches to manipulating gene editing outcomes.
Topics: Animals; Chromosome Breakage; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; Humans; Mammals; Mutagenesis
PubMed: 35926296
DOI: 10.1016/j.dnarep.2022.103380 -
Methods in Molecular Biology (Clifton,... 2022R-loop is a three-stranded chromatin structure, comprising one single-stranded DNA and another DNA:RNA hybrid strand, plays various and essential biological functions in...
R-loop is a three-stranded chromatin structure, comprising one single-stranded DNA and another DNA:RNA hybrid strand, plays various and essential biological functions in many organisms. Developing a precise, efficient, faithful, and unbiased genome-wide R-loop detection method with extensive adaptability in all organisms is at the top priority for R-loop biology. Here, we provide a straightforward and highly efficient protocol for genome-wide strand-specific R-loop profiling in various organisms. In brief, genomic DNA is extracted and fragmented by the cocktail of restriction enzymes, and then the DNA:RNA hybrids are immunoprecipitated, following by the single-stranded DNA adaptor ligation and next-generation sequencing (named as ssDRIP-seq). Coupling with a straightforward and step-by-step bioinformatic pipeline, this method can provide high resolution and comprehensive strand-specific information for R-loop formation. ssDRIP-seq has been successfully applied for detecting R-loops from prokaryotes such as E. coli, to eukaryotes such as S. cerevisiae, mammalian cell culture and tissues, as well as plants Arabidopsis and rice, with high reproducibility and sensitivity.
Topics: Animals; DNA; DNA, Single-Stranded; Escherichia coli; Mammals; R-Loop Structures; RNA; Reproducibility of Results; Saccharomyces cerevisiae
PubMed: 35704209
DOI: 10.1007/978-1-0716-2477-7_29 -
Environmental and Molecular Mutagenesis 2024As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a...
As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a method reliably assessing DNA ligation capacity in protein extracts from murine tissues was aimed to establish. To optimize applicability, the use of radioactively labeled substrates was avoided and replaced by fluorescently labeled oligonucleotides. Briefly, tissue extracts were incubated with those complementary oligonucleotides so that in an ensuing gel electrophoresis ligated strands could be separated from unconnected molecules. Originally, the method was intended for use in cerebellum tissue to further elucidate possible mechanisms of neurodegenerative diseases. However, due to its inhomogeneous anatomy, DNA ligation efficiency varied strongly between different cerebellar areas, illuminating the established assay to be suitable only for homogenous organs. Thus, for murine liver tissue sufficient intra- and interday repeatability was shown during validation. In further experiments, the established assay was applied to an animal study comprising young and old (24 and 110 weeks) mice which showed that DNA ligation efficiency was affected by neither sex nor age. Finally, the impact of in vitro addition of the trace elements copper, iron, and zinc on DNA ligation in tissue extracts was investigated. While all three metals inhibited DNA ligation, variations in their potency became evident. In conclusion, the established method can be reliably used for investigation of DNA ligation efficiency in homogenous murine tissues.
Topics: Animals; Mice; DNA; Male; Female; Liver; Cerebellum; Mice, Inbred C57BL; DNA Ligases; DNA Repair
PubMed: 38767089
DOI: 10.1002/em.22602 -
Protein and Peptide Letters 2022Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research...
BACKGROUND
Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source.
OBJECTIVE
The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases.
METHODS
We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions.
RESULTS
In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates.
CONCLUSION
Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.
Topics: DNA Ligases; DNA, Recombinant; Escherichia coli; Polyethylene Glycols; Proteins
PubMed: 35657285
DOI: 10.2174/0929866529666220426122432 -
Angewandte Chemie (International Ed. in... Apr 2023Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA...
Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA nanostructures. However, it is challenging to prepare such ssDNA circles, particularly when the DNA size becomes very small (e.g. a 20 nucleotide (nt) long ssDNA circle). Often, such short ssDNA dominantly form concatemers (either linear or circular) due to intermolecular ligation, instead of forming monomeric ssDNA circles by intramolecular ligation. Herein, a simple method to overcome this problem by designing the complementary linker molecules is reported. It is demonstrated that ssDNA, as short as 16 nts, can be enzymatically ligated (by the commonly used T4 DNA ligase) into monomeric ssDNA circles at high concentration (100 μM) with high yield (97 %). This method does not require any special sequence, thus, it is expected to be generally applicable. The experimental protocol is identical to regular DNA ligation, thus, is expected to be user friendly for general chemists and biologists.
Topics: DNA, Single-Stranded; DNA; Nucleotides; Nanostructures; DNA Ligases; Nucleic Acid Amplification Techniques; DNA, Circular
PubMed: 36652628
DOI: 10.1002/anie.202218443 -
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 the American Chemical Society Oct 2019Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has...
Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has been hindered by the highly constrained conditions for DNA crystals to be stable. Here we report a method to prepare robust 3D DNA crystals by postassembly ligation to remove this constraint. Specifically, sticky ends at crystal contacts were enzymatically ligated, and the covalent bonds significantly enhanced crystal stability, e.g., being stable at 65 °C. This method also enabled the fabrication of DNA crystals with complex architectures including crystal shell, core-shell, and matryoshka dolls. Furthermore, we have demonstrated the applications of the robust DNA crystals in biocatalysis and protein entrapment. Our study removes one key obstacle for the applications of DNA crystals and offers many new opportunities in DNA nanotechnology.
Topics: Crystallization; DNA; DNA Ligases; Microscopy, Electron, Transmission; Nanotechnology; Nucleic Acid Conformation; Stress, Mechanical; X-Ray Diffraction
PubMed: 31553173
DOI: 10.1021/jacs.9b06613 -
The Journal of Biological Chemistry 2021DNA ligase I (LIG1) completes the base excision repair (BER) pathway at the last nick-sealing step after DNA polymerase (pol) β gap-filling DNA synthesis. However, the...
DNA ligase I (LIG1) completes the base excision repair (BER) pathway at the last nick-sealing step after DNA polymerase (pol) β gap-filling DNA synthesis. However, the mechanism by which LIG1 fidelity mediates the faithful substrate-product channeling and ligation of repair intermediates at the final steps of the BER pathway remains unclear. We previously reported that pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion confounds LIG1, leading to the formation of ligation failure products with a 5'-adenylate block. Here, using reconstituted BER assays in vitro, we report the mutagenic ligation of pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion products and an inefficient ligation of pol β Watson-Crick-like dG:T mismatch insertion by the LIG1 mutant with a perturbed fidelity (E346A/E592A). Moreover, our results reveal that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches is governed by mutations at both E346 and E592 residues. Finally, we found that aprataxin and flap endonuclease 1, as compensatory DNA-end processing enzymes, can remove the 5'-adenylate block from the abortive ligation products harboring 3'-8-oxodG or the 12 possible noncanonical base pairs. These findings contribute to the understanding of the role of LIG1 as an important determinant in faithful BER and how a multiprotein complex (LIG1, pol β, aprataxin, and flap endonuclease 1) can coordinate to prevent the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway.
Topics: DNA; DNA Ligase ATP; DNA Polymerase beta; DNA Repair; DNA Replication; Flap Endonucleases; Humans; Mutagenesis; Mutagens; Mutation; Nucleotides; Oxidation-Reduction
PubMed: 33600799
DOI: 10.1016/j.jbc.2021.100427 -
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