Showing papers on "genomic DNA published in 2020"

Quantum scale organic semiconductors for SERS detection of DNA methylation and gene expression

Abstract: Cancer stem cells (CSC) can be identified by modifications in their genomic DNA. Here, we report a concept of precisely shrinking an organic semiconductor surface-enhanced Raman scattering (SERS) probe to quantum size, for investigating the epigenetic profile of CSC. The probe is used for tag-free genomic DNA detection, an approach towards the advancement of single-molecule DNA detection. The sensor detected structural, molecular and gene expression aberrations of genomic DNA in femtomolar concentration simultaneously in a single test. In addition to pointing out the divergences in genomic DNA of cancerous and non-cancerous cells, the quantum scale organic semiconductor was able to trace the expression of two genes which are frequently used as CSC markers. The quantum scale organic semiconductor holds the potential to be a new tool for label-free, ultra-sensitive multiplexed genomic analysis. The low detection sensitivity of organic semiconductors has limited their use in biomedical surface-enhanced Raman scattering applications. Here, the authors use quantum scale organic semiconductors and show detection of genomic DNA methylation as well as gene expression.

Programming Switchable Transcription of Topologically Constrained DNA

Abstract: Genomic DNA is compacted via chromatin condensation in mammalian cells, and transcription of such topologically constrained DNA to messenger RNA is under strict spatiotemporal regulation. Nevertheless, control of DNA topology has been poorly explored in in vitro transcription and gene transfection. Here we report the construction of topologically ordered (TO-) prokaryotic genes composed of linear DNA templates appended with a T7 promoter sequence with the use of DNA self-assembly. We find that TO-DNA maintains the transcription activity whereas the activity is critically dependent on the configuration of the T7 promoter in a folded DNA nanostructure. By prescribing the position and the intactness of the T7 promoter, we can dynamically activate or repress transcription in response to specific DNA key strands in a Boolean logic manner. Bioorthogonal switchable transcription is realized with the insertion of multiple genes in a TO-DNA. Further, implementing TO-DNA in living bacteria leads to switchable transcription of fluorescent RNA aptamers for light-up cell imaging. Hence, the design of TO-DNAs provides a means for shape-dependent gene delivery, enriching the toolbox of genetic engineering and synthetic biology.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer.

Abstract: Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

Single-cell sequencing of genomic DNA resolves sub-clonal heterogeneity in a melanoma cell line

Abstract: We performed shallow single-cell sequencing of genomic DNA across 1475 cells from a cell-line, COLO829, to resolve overall complexity and clonality This melanoma tumor-line has been previously characterized by multiple technologies and is a benchmark for evaluating somatic alterations In some of these studies, COLO829 has shown conflicting and/or indeterminate copy number and, thus, single-cell sequencing provides a tool for gaining insight Following shallow single-cell sequencing, we first identified at least four major sub-clones by discriminant analysis of principal components of single-cell copy number data Based on clustering, break-point and loss of heterozygosity analysis of aggregated data from sub-clones, we identified distinct hallmark events that were validated within bulk sequencing and spectral karyotyping In summary, COLO829 exhibits a classical Dutrillaux’s monosomic/trisomic pattern of karyotype evolution with endoreduplication, where consistent sub-clones emerge from the loss/gain of abnormal chromosomes Overall, our results demonstrate how shallow copy number profiling can uncover hidden biological insights Through shallow single-cell sequencing of genomic DNA followed by clustering analysis, Velazquez-Villarreal et al reveal sub-clones of the melanoma cell line COLO829 and further identify and validate chromosome translocations and copy number changes This study illustrates how copy number variation analysis can provide insights into cancer cell heterogeneity

Structures and stability of simple DNA repeats from bacteria

Abstract: DNA is a fundamentally important molecule for all cellular organisms due to its biological role as the store of hereditary, genetic information. On the one hand, genomic DNA is very stable, both in chemical and biological contexts, and this assists its genetic functions. On the other hand, it is also a dynamic molecule, and constant changes in its structure and sequence drive many biological processes, including adaptation and evolution of organisms. DNA genomes contain significant amounts of repetitive sequences, which have divergent functions in the complex processes that involve DNA, including replication, recombination, repair, and transcription. Through their involvement in these processes, repetitive DNA sequences influence the genetic instability and evolution of DNA molecules and they are located non-randomly in all genomes. Mechanisms that influence such genetic instability have been studied in many organisms, including within human genomes where they are linked to various human diseases. Here, we review our understanding of short, simple DNA repeats across a diverse range of bacteria, comparing the prevalence of repetitive DNA sequences in different genomes. We describe the range of DNA structures that have been observed in such repeats, focusing on their propensity to form local, non-B-DNA structures. Finally, we discuss the biological significance of such unusual DNA structures and relate this to studies where the impacts of DNA metabolism on genetic stability are linked to human diseases. Overall, we show that simple DNA repeats in bacteria serve as excellent and tractable experimental models for biochemical studies of their cellular functions and influences.

Assessment of DNA Epigenetic Modifications.

Abstract: DNA molecules utilize adenine, thymine, cytosine, and guanine for coding genetic information. In addition to these four canonical nucleobases, DNA molecules also contain a variety of modified nucleobases that can control and regulate gene expression and chromosome structure. Elucidating the functions of DNA modifications relies on the sensitive detection, accurate quantification, and genome-wide mapping of these modifications in genomic DNA. The significant advances of techniques and methods in recent years have enabled the discovery and functional studies of a number of new modifications in DNA in both prokaryotes and eukaryotes. Mass spectrometry-based methods for analyzing DNA modifications have substantially advanced over the past decade, which has greatly stimulated the research of DNA epigenetic modifications. The emergence of next-generation sequencing technology provides complementary methodology to enable genome-wide mapping of modifications, which is very important to reveal the biological roles of DNA modifications. This perspective highlights the recent methodologies for the assessment of DNA modifications with focus on mass spectrometry and sequencing analytical strategies.

Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Abstract: One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

Genomic methods for measuring DNA replication dynamics

Abstract: Genomic DNA replicates according to a defined temporal program in which early-replicating loci are associated with open chromatin, higher gene density, and increased gene expression levels, while late-replicating loci tend to be heterochromatic and show higher rates of genomic instability. The ability to measure DNA replication dynamics at genome scale has proven crucial for understanding the mechanisms and cellular consequences of DNA replication timing. Several methods, such as quantification of nucleotide analog incorporation and DNA copy number analyses, can accurately reconstruct the genomic replication timing profiles of various species and cell types. More recent developments have expanded the DNA replication genomic toolkit to assays that directly measure the activity of replication origins, while single-cell replication timing assays are beginning to reveal a new level of replication timing regulation. The combination of these methods, applied on a genomic scale and in multiple biological systems, promises to resolve many open questions and lead to a holistic understanding of how eukaryotic cells replicate their genomes accurately and efficiently.

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

Abstract: With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

Unraveling epigenomic abnormality in azoospermic human males by WGBS, RNA-Seq, and transcriptome profiling analyses.

Abstract: To determine associations between genomic DNA methylation in testicular cells and azoospermia in human males. This was a case-control study investigating the differences and conservations in DNA methylation, genome-wide DNA methylation, and bulk RNA-Seq for transcriptome profiling using testicular biopsy tissues from NOA and OA patients. Differential methylation and different conserved methylation regions associated with azoospermia were identified by comparing genomic DNA methylation of testicular seminiferous cells derived from NOA and OA patients. The genome methylation modification of testicular cells from NOA patients was disordered, and the reproductive-related gene expression was significantly different. Our findings not only provide valuable knowledge of human spermatogenesis but also paved the way for the identification of genes/proteins involved in male germ cell development. The approach presented in this report provides a powerful tool to identify responsible biomolecules, and/or cellular changes (e.g., epigenetic abnormality) that induce male reproductive dysfunction such as OA and NOA.

ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing

Abstract: Identifying nuclease-induced double-stranded breaks in DNA on a genome-wide scale is critical for assessing the safety and efficacy of genome editing therapies. We previously demonstrated that after administering adeno-associated viral (AAV) vector-mediated genome-editing strategies in vivo, vector sequences integrated into the host organism’s genomic DNA at double-stranded breaks. Thus, identifying the genomic location of inserted AAV sequences would enable us to identify DSB events, mainly derived from the nuclease on- and off-target activity. Here, we developed a next-generation sequencing assay that detects insertions of specific AAV vector sequences called inverted terminal repeats (ITRs). This assay, ITR-Seq, enables us to identify off-target nuclease activity in vivo. Using ITR-Seq, we analyzed liver DNA samples of rhesus macaques treated with AAV vectors expressing a meganuclease. We found dose-dependent off-target activity and reductions in off-target events induced by further meganuclease development. In mice, we identified the genomic locations of ITR integration after treatment with Cas9 nucleases and their corresponding single-guide RNAs. In sum, ITR-Seq is a powerful method for identifying off-target sequences induced by AAV vector-delivered genome-editing nucleases. ITR-Seq will help us understand the specificity and efficacy of different genome-editing nucleases in animal models and clinical studies. This information can help enhance the safety profile of gene-editing therapies.

Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.

Abstract: Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

Tomato Yellow Leaf Curl Virus V2 Protein Plays a Critical Role in the Nuclear Export of V1 Protein and Viral Systemic Infection.

Abstract: Geminiviruses are an important group of circular, single-stranded DNA viruses that cause devastating diseases in crops. Geminiviruses replicate their genomic DNA in the nucleus and the newly synthesized viral DNA is subsequently transported to the cytoplasm for further cell-to-cell and long-distance movement to establish systemic infection. Thus, nucleocytoplasmic transportation is crucial for successful infection by geminiviruses. For Tomato yellow leaf curl virus (TYLCV), the V1 protein is known to bind and shuttle viral genomic DNA, however, the role of the V2 protein in this process is still unclear. Here, we report that the V1 protein is primarily localized in the nucleus when expressed but the nucleus-localized V1 protein dramatically decreases when co-expressed with V2 protein. Moreover, the V2-facilitated nuclear export of V1 protein depends on host exportin-α and a specific V1-V2 interaction. Chemical inhibition of exportin-α or a substitution at cysteine 85 of the V2 protein, which abolishes the V1-V2 interaction, blocks redistribution of the V1 protein to the perinuclear region and the cytoplasm. When the V2C85S mutation is incorporated into a TYLCV infectious clone, the TYLCV-C85S causes delayed onset of very mild symptoms compared to wild-type TYLCV, suggesting that the V1-V2 interaction and, thus, the V2-mediated nuclear export of the V1 protein is crucial for viral spread and systemic infection. Our data point to a critical role of the V2 protein in promoting the nuclear export of the V1 protein and viral systemic infection, likely by promoting V1 protein-mediated nucleocytoplasmic transportation of TYLCV genomic DNA.

Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M.

Abstract: Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4–5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1–2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues. Here, the authors provide detailed experimental and analytical procedures for Hi-M, a method that enables simultaneous imaging of 3D genome folding and RNA localization in single cells.

Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing.

Abstract: Replication timing (RT) domains are stable units of chromosome structure that are regulated in the context of development and disease. Conventional genome-wide RT mapping methods require many S-phase cells for either the effective enrichment of replicating DNA through bromodeoxyuridine (BrdU) immunoprecipitation or the determination of copy-number differences during S-phase, which precludes their application to non-abundant cell types and single cells. Here, we provide a simple, cost-effective, and robust protocol for single-cell DNA replication sequencing (scRepli-seq). The scRepli-seq methodology relies on whole-genome amplification (WGA) of genomic DNA (gDNA) from single S-phase cells and next-generation sequencing (NGS)-based determination of copy-number differences that arise between replicated and unreplicated DNA. Haplotype-resolved scRepli-seq, which distinguishes pairs of homologous chromosomes within a single cell, is feasible by using single-nucleotide polymorphism (SNP)/indel information. We also provide computational pipelines for quality control, normalization, and binarization of the scRepli-seq data. The experimental portion of this protocol (before sequencing) takes 3 d. This protocol describes experimental and computational procedures for obtaining genome-wide DNA replication timing maps based on copy-number differences derived from whole-genome amplification and next-generation sequencing of genomic DNA from single S-phase cells.

First record of a tandem-repeat region within the mitochondrial genome of Clonorchis sinensis using a long-read sequencing approach.

Abstract: Background Mitochondrial genomes provide useful genetic markers for systematic and population genetic studies of parasitic helminths. Although many such genome sequences have been published and deposited in public databases, there is evidence that some of them are incomplete relating to an inability of conventional techniques to reliably sequence non-coding (repetitive) regions. In the present study, we characterise the complete mitochondrial genome—including the long, non-coding region—of the carcinogenic Chinese liver fluke, Clonorchis sinensis, using long-read sequencing. Methods The mitochondrial genome was sequenced from total high molecular-weight genomic DNA isolated from a pool of 100 adult worms of C. sinensis using the MinION sequencing platform (Oxford Nanopore Technologies), and assembled and annotated using an informatic approach. Results From > 93,500 long-reads, we assembled a 18,304 bp-mitochondrial genome for C. sinensis. Within this genome we identified a novel non-coding region of 4,549 bp containing six tandem-repetitive units of 719–809 bp each. Given that genomic DNA from pooled worms was used for sequencing, some variability in length/sequence in this tandem-repetitive region was detectable, reflecting population variation. Conclusions For C. sinensis, we report the complete mitochondrial genome, which includes a long (> 4.5 kb) tandem-repetitive region. The discovery of this non-coding region using a nanopore-sequencing/informatic approach now paves the way to investigating the nature and extent of length/sequence variation in this region within and among individual worms, both within and among C. sinensis populations, and to exploring whether this region has a functional role in the regulation of replication and transcription, akin to the mitochondrial control region in mammals. Although applied to C. sinensis, the technological approach established here should be broadly applicable to characterise complex tandem-repetitive or homo-polymeric regions in the mitochondrial genomes of a wide range of taxa.

Complete Genome and Plasmids Sequences of a Clinical Proteus mirabilis Isolate Producing Plasmid Mediated NDM-1 from Italy

Abstract: Background: The spread of carbapenemase genes, such as blaNDM-1, in Proteus mirabilis poses a public health threat. The aim of the study was to characterize the genome and plasmids sequences of an NDM-1-positive strain (IBCRE14), which was isolated in 2019 from a catheterized patient hospitalized in Italy. Methods: Whole genome sequencing (WGS) of IBCRE14 was performed on extracted genomic DNA using Sequel I platform. Genome assembly was performed using "Microbial Assembly". Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases from the Center for Genomic Epidemiology. Results: IBCRE14 had a genome size of 4,018,329 bp and harboured genes coding for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ), and trimethoprim (dfrA1). A large plasmid (pIB_NDM_1) harboured antibiotic resistance genes against sulphonamide (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr-2), aminoglycosides (aadA1, aph3-VI), and beta-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) harboured a qnrD1 gene coding for quinolone resistance. Conclusion: The ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could both acquire more resistance genes and easily disseminate. To our knowledge, this is the first report on an untypable plasmid harbouring blaNDM-1 in P. mirabilis, in Italy.

The structure of APOBEC1 and insights into its RNA and DNA substrate selectivity.

Abstract: APOBEC1 (APO1), a member of AID/APOBEC nucleic acid cytosine deaminase family, can edit apolipoprotein B mRNA to regulate cholesterol metabolism. This APO1 RNA editing activity requires a cellular cofactor to achieve tight regulation. However, no cofactors are required for deamination on DNA by APO1 and other AID/APOBEC members, and aberrant deamination on genomic DNA by AID/APOBEC deaminases has been linked to cancer. Here, we present the crystal structure of APO1, which reveals a typical APOBEC deaminase core structure, plus a unique well-folded C-terminal domain that is highly hydrophobic. This APO1 C-terminal hydrophobic domain (A1HD) interacts to form a stable dimer mainly through hydrophobic interactions within the dimer interface to create a four-stranded β-sheet positively charged surface. Structure-guided mutagenesis within this and other regions of APO1 clarified the importance of the A1HD in directing RNA and cofactor interactions, providing insights into the structural basis of selectivity on DNA or RNA substrates.

A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus.

Abstract: Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.4 kb fragment from the human genome. Through a streamlined process of substrate-linked directed evolution we generated two separate recombinases that, when expressed together, act as a heterodimer to delete a human genomic sequence from chromosome 7. Our data indicates that designer-recombinases can be generated in a manageable timeframe for precision genome editing. A large-scale bioinformatics analysis suggests that around 13% of all human protein-coding genes could be targetable by dual designer-recombinase induced genomic deletion (dDRiGD). We propose that heterospecific designer-recombinases, which work independently of the host DNA repair machinery, represent an efficient and safe alternative to nuclease-based genome editing technologies.

Inhibition of topoisomerase IIA (Top2α) induces telomeric DNA damage and T cell dysfunction during chronic viral infection

Abstract: T cells play a critical role in controlling viral infection; however, the mechanisms regulating their responses remain incompletely understood. Here, we investigated the role of topoisomerase IIA (Top2α, an enzyme that is essential in resolving entangled DNA strands during replication) in telomeric DNA damage and T cell dysfunction during viral infection. We demonstrated that T cells derived from patients with chronic viral (HBV, HCV, and HIV) infection had lower Top2α protein levels and enzymatic activity, along with an accumulation of the Top2α cleavage complex (Top2cc) in genomic DNA. In addition, T cells from virally infected subjects with lower Top2α levels were vulnerable to Top2α inhibitor-induced cell apoptosis, indicating an important role for Top2α in preventing DNA topological disruption and cell death. Using Top2α inhibitor (ICRF193 or Etoposide)-treated primary T cells as a model, we demonstrated that disrupting the DNA topology promoted DNA damage and T cell apoptosis via Top2cc accumulation that is associated with protein-DNA breaks (PDB) at genomic DNA. Disruption of the DNA topology was likely due to diminished expression of tyrosyl-DNA phosphodiesterase 2 (TDP2), which was inhibited in T cells in vitro by Top2α inhibitor and in vivo by chronic viral infection. These results suggest that immune-evasive viruses (HBV, HCV, and HIV) can disrupt T cell DNA topology as a mechanism of dysregulating host immunity and establishing chronic infection. Thus, restoring the DNA topologic machinery may serve as a novel strategy to protect T cells from unwanted DNA damage and to maintain immune competence.

High‐throughput sequencing of T‐cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies

Abstract: Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.