DNA Molecules Research Articles

Characterization and genomic analysis phage PmP19, a novel Proteus mirabilis phage with a broad host range.

A lytic Proteus mirabilis phage, PmP19, was isolated from sewage on a farm. PmP19 has an icosahedral head (60 ± 3 nm in diameter) and a short tail (15 ± 2 nm long). Its genome, a linear, double-stranded DNA molecule 44,305 bp in length with an average GC content of 51.93%, has 52 putative open reading frames (ORFs). BLASTn comparisons and phylogenetic analysis revealed a close relationship between Pmp19 and Klebsiella phage vB_KpnP_ZK1. Bioinformatic analysis revealed that PmP19 belongs to the phage subfamily Molineuxvirinae.

Transfer learning Bayesian optimization for competitor DNA molecule design for use in diagnostic assays.

With the rise in engineered biomolecular devices, there is an increased need for tailor-made biological sequences. Often, many similar biological sequences need to be made for a specific application meaning numerous, sometimes prohibitively expensive, lab experiments are necessary for their optimization. This paper presents a transfer learning design of experiments workflow to make this development feasible. By combining a transfer learning surrogate model with Bayesian optimization, we show how the total number of experiments can be reduced by sharing information between optimization tasks. We demonstrate the reduction in the number of experiments using data from the development of DNA competitors for use in an amplification-based diagnostic assay. We use cross-validation to compare the predictive accuracy of different transfer learning models, and then compare the performance of the models for both single objective and penalized optimization tasks.

GATA1 transcription factor targets the gene expression of B19 virus in HEK293 cell line

Backgrond/Aim: B19 virus (B19V) is a single strand DNA virus that has specific tropism to erythroid progenitor cells (EPCs). The virus enters to the cells via P antigen and coreseptors and induces infection and cell apoptosis. GATA1 has a high expression in EPC and is a critical transcription factor for the cells development and differentiation. As human EPCs are the main target of the virus infection that have high expression of GATA-1 as the critical transcription factor, the aim of this study was to investigate the effect of GATA1 cotransfection with B19V genome on expression of the viral mRNAs in HEK293 as non-permissive cell line to the virus that had no mRNA expression of GATA-1. Methods: HEK293 cells were transfected with pHI0 plasmid containing B19V genome and the plasmid of GATA1 genome. The quantity of B19V mRNAs (NS1, 7.5 kDa, 11 kDa) expression was evaluated after 24h of transfection. Results: The results showed a statistically significant increase in fold change expression of (NS1 ∽ 12.3, VP1 ∽ 27.6, 11kb protein ∽ 38) in cotransfected cells with GATA1 and B19 plasmids compare to control group (P<0.05). Conclusion: This research showed transfected cells with GATA1had elevation in expression of the B19V genes mRNAs in a non-permissive cell. This result may show a role of GATA1 as a critical transcription factor in support of the virus infection in EPCs. This suggests that GATA1 may potentially sport B19V replication or gene expression.

The Smc5/6 complex counteracts R-loop formation at highly transcribed genes in cooperation with RNase H2.

The R-loop is a common transcriptional by-product that consists of an RNA-DNA duplex joined to a displaced strand of genomic DNA. While the effects of R-loops on health and disease are well established, there is still an incomplete understanding of the cellular processes responsible for their removal from eukaryotic genomes. Here, we show that a core regulator of chromosome architecture -the Smc5/6 complex- plays a crucial role in the removal of R-loop structures formed during gene transcription. Consistent with this, budding yeast mutants defective in the Smc5/6 complex and enzymes involved in R-loop resolution show strong synthetic interactions and accumulate high levels of RNA-DNA hybrid structures in their chromosomes. Importantly, we demonstrate that the Smc5/6 complex acts on specific types of RNA-DNA hybrid structures in vivo and promotes R-loop degradation by the RNase H2 enzyme in vitro. Collectively, our results reveal a crucial role for the Smc5/6 complex in the removal of toxic R-loops formed at highly transcribed genes and telomeres.

Causes and consequences of DNA double-stranded breaks in cardiovascular disease.

The genome, whose stability is essential for survival, is incessantly exposed to internal and external stressors, which introduce an estimated 104 to 105 lesions, such as oxidation, in the nuclear genome of each mammalian cell each day. A delicate homeostatic balance between the generation and repair of DNA lesions maintains genomic stability. To initiate transcription, DNA strands unwind to form a transcription bubble and provide a template for the RNA polymerase II (RNAPII) complex to synthesize nascent RNA. The process generates DNA supercoils and introduces torsional stress. To enable RNAPII processing, the supercoils are released by topoisomerases by introducing strand breaks, including double-stranded breaks (DSBs). Thus, DSBs are intrinsic genomic features of gene expression. The breaks are quickly repaired upon processing of the transcription. DNA lesions and damaged proteins involved in transcription could impede the integrity and efficiency of RNAPII processing. The impediment, which is referred to as transcription stress, not only could lead to the generation of aberrant RNA species but also the accumulation of DSBs. The latter is particularly the case when topoisomerase processing and/or the repair mechanisms are compromised. The DSBs activate the DNA damage response (DDR) pathways to repair the damaged DNA and/or impose cell cycle arrest and cell death. In addition, the release of DSBs into the cytosol activates the cytosolic DNA-sensing proteins (CDSPs), which along with the nuclear DDR pathways induce the expression of senescence-associated secretory phenotype (SASP), cell cycle arrest, senescence, cell death, inflammation, and aging. The primary stimulus in hereditary cardiomyopathies is a mutation(s) in genes encoding the protein constituents of cardiac myocytes; however, the phenotype is the consequence of intertwined complex interactions among numerous stressors and the causal mutation(s). Increased internal DNA stressors, such as oxidation, alkylation, and cross-linking, are expected to be common in pathological conditions, including in hereditary cardiomyopathies. In addition, dysregulation of gene expression also imposes transcriptional stress and collectively with other stressors provokes the generation of DSBs. In addition, the depletion of nicotinamide adenine dinucleotide (NAD), which occurs in pathological conditions, impairs the repair mechanism and further facilitates the accumulation of DSBs. Because DSBs activate the DDR pathways, they are expected to contribute to the pathogenesis of cardiomyopathies. Thus, interventions to reduce the generation of DSBs, enhance their repair, and block the deleterious DDR pathways would be expected to impart salubrious effects not only in pathological states, as in hereditary cardiomyopathies but also aging.

Effect of Ultrahigh Dose Rate on Biomolecular Radiation Damage.

Dose rate is one of the important parameters in radiation-induced biomolecular damage. The effects of dose rate have been known to modify radiation toxicity in biological systems. The rate and extent of sublethal DNA damage (e.g., base damage and single-strand breaks) repair and those of cell proliferation have been manifested by dose rate. However, the recent preclinical application of ultrahigh dose rate [(UHDR) ca. 40 Gy/s and higher] radiation modalities have been shown to lower the type and extent of radiation damage to biological systems. At these UHDR, radiation-induced physicochemical and chemical processes are expected to differ from those observed after irradiation at conventional dose rates (CONV). It is unclear whether these UHDR conditions can affect the quality (type) and quantity (extent) of biomolecular damage such as DNA lesions. Here, we comparatively study the influence of indirect effects of CONV and UHDR on the formation of DNA strand breaks and clustered damage including densely accumulated lesions in an aerated and an anoxic dilute aqueous solution of a plasmid DNA model under low and high hydroxyl radical (•OH) scavenging conditions. Aqueous solutions of purified supercoiled plasmid DNA (pUC19) were prepared in either air- or nitrogen-saturated conditions, with Tris buffer added as the radiation-produced •OH scavenger at low and high scavenging capacities. These DNA samples were irradiated using kV X-ray systems at CONV (0.1 Gy/s) and high dose rate (HDR, 25 Gy/s) as well as UHDR (55 and 125 Gy/s) under different scavenging and environmental conditions. DNA lesions including strand breaks and clustered damage including densely accumulated lesions were quantified by gel electrophoresis and the yields of these lesions were calculated from the dose-response curve. Non-DSB clustered damage including densely accumulated lesions were evaluated by treating DNAs using bacterial endonuclease enzymes (Fpg and Nth) prior to gel electrophoresis. UHDR of 55 and 125 Gy/s induced lower amounts of both isolated strand breaks and clustered DNA damage including densely accumulated lesions at doses >40 Gy in the presence of oxygen, compared to the abundance of these lesions induced by 0.1 and 25 Gy/s irradiation under the same dose conditions. Overall, the strand break and clustered damage including densely accumulated lesions yields decreased by factors of 1.3-3.5 after UHDR. We did not observe these differences either via •OH scavenging or by removing oxygen from the solution. In addition, our results point out that the inter-track recombination reactions did not contribute to the observed dose-rate effects on DNA damage. The effects of dose rate on DNA damage are highly dependent on the total dose, as expected, but also on the •OH scavenging capacity that is employed in the aqueous DNA solutions. These important variables may be relevant in biological systems as well. On a practical level, our in vitro plasmid DNA model, which permits to precisely vary the scavenging capacity and gassing conditions (air saturated vs. N2 saturated) can help to differentiate dose-rate effects on biomolecular damage. Our results indicate that the radical-radical reactions are important in understanding the dose-rate effect on DNA damage.

Diverse new plasmid structures and antimicrobial resistance in strains isolated from perianal abscess patients.

Plasmids, the most important and versatile bacterial extrachromosomal DNA Molecules, has have been a center central topic for bacterial genetics and biology. However, the inability of short-read high-throughput sequencing methods to reliably assemble plasmids makes it difficult to investigate the diversity of plasmid structures and functions. In this work, we used the long-read Nanopore sequencing method to address this issue, by producing high quality whole genome sequences of 33 bacterial strains from 11 perianal abscess-suffering patients. Successful high quality assemblies were generated with this method, including 20 perfect assemblies out of 33 genomes. A total of 47 plasmids were identified from the bacterial strains, including 12 unique, newly identified, high quality circular plasmids. These plasmids were further subject to structural analysis, leading to the finding of significant diversification from previously known plasmids, suggesting the diversity of plasmid structure and function. Particularly, two mcr-10.1-harboring conjugative plasmids were found from Citrobacter portucalensis and Enterobacter kobei, which were not previously reported. This works shows the feasibility of using long-read sequencing to identify plasmids, and the high diversity of plasmid structure and function that awaits further surveillance.

Efficient Multidriven Strand Displacement Reaction for Biosensing.

A key challenge for achieving high-efficient DNA strand displacement reaction (SDR) with existing technologies is the inferior kinetic performance due to the alternately cumbersome conjunction and dissociation of dsDNA. In this work, a novel multidriven SDR collaborated by toehold initiator, strand towing, and click chemistry is engineered. The invasion strand (O) endows the hybridization with a basal strand (M) in dsDNA for releasing a displacement strand (P), which can be significantly boosted by the towing of a helper strand and impetus from the click reaction. Accordingly, the hybridization rate and dissociation extent of P can be largely improved and showed a desiring displacement rate close to 6-fold compared with the traditional method, providing a newly high-efficient SDR strategy for potential application in biosensing, clinical diagnostics, and DNA nanotechnology. In view of this, a practical biosensing platform by combining the multidriven SDR (MSDR) with waste-free DNA multi-cycle amplification is constructed for the rapid and ultrasensitive electrochemical detection of cancer-related miRNA-21. The substantial output DNA as an invasion strand (O) from target-triggered waste-free DNA multicycle can high-efficiently release a signal probe (Fc)-labeled displacement strand (P) on an electrode by using the proposed MSDR, obtaining a low detection limit below 106.8 aM.

Absence of genotoxicity following pulmonary exposure to metal oxides of copper, tin, aluminum, zinc, and titanium in mice.

Inhalation of nanosized metal oxides may occur at the workplace. Thus, information on potential hazardous effects is needed for risk assessment. We report an investigation of the genotoxic potential of different metal oxide nanomaterials. Acellular and intracellular reactive oxygen species (ROS) production were determined for all the studied nanomaterials. Moreover, mice were exposed by intratracheal instillation to copper oxide (CuO) at 2, 6, and 12 μg/mouse, tin oxide (SnO2) at 54 and 162 μg/mouse, aluminum oxide (Al2O3) at 18 and 54 μg/mouse, zinc oxide (ZnO) at 0.7 and 2 μg/mouse, titanium dioxide (TiO2) and the benchmark carbon black at 162 μg/mouse. The doses were selected based on pilot studies. Post-exposure time points were 1 or 28 days. Genotoxicity, assessed as DNA strand breaks by the comet assay, was measured in lung and liver tissue. The acellular and intracellular ROS measurements were fairly consistent. The CuO and the carbon black bench mark particle were potent ROS generators in both assays, followed by TiO2. Al2O3, ZnO, and SnO2 generated low levels of ROS. We detected no increased genotoxicity in this study using occupationally relevant dose levels of metal oxide nanomaterials after pulmonary exposure in mice, except for a slight increase in DNA damage in liver tissue at the highest dose of CuO. The present data add to the body of evidence for risk assessment of these metal oxides.

Comparative analysis of the whole mitochondrial genomes of four species in sect. Chrysantha (Camellia L.), endemic taxa in China

BackgroundThe sect. Chrysantha Chang of plants with yellow flowers of Camellia species as the “Queen of the Tea Family”, most of these species are narrowly distributed endemics of China and are currently listed Grde-II in National Key Protected Wild Plant of China. They are commercially important plants with horticultural medicinal and scientific research value. However, the study of the sect. Chrysantha species genetics are still in its infancy, to date, the mitochondrial genome in sect. Chrysantha has been still unexplored.ResultsIn this study, we provide a comprehensive assembly and annotation of the mitochondrial genomes for four species within the sect. Chrysantha. The results showed that the mitochondrial genomes were composed of closed-loop DNA molecules with sizes ranging from 850,836 bp (C. nitidissima) to 1,098,121 bp (C. tianeensis) with GC content of 45.71–45.78% and contained 48–58 genes, including 28–37 protein-coding genes, 17–20 tRNA genes and 2 rRNA genes. We also examined codon usage, sequence repeats, RNA editing and selective pressure in the four species. Then, we performed a comprehensive comparison of their basic structures, GC contents, codon preferences, repetitive sequences, RNA editing sites, Ka/Ks ratios, haplotypes, and RNA editing sites. The results showed that these plants differ little in gene type and number. C. nitidissima has the greatest variety of genes, while C. tianeensis has the greatest loss of genes. The Ka/Ks values of the atp6 gene in all four plants were greater than 1, indicating positive selection. And the codons ending in A and T were highly used. In addition, the RNA editing sites differed greatly in number, type, location, and efficiency. Twelve, six, five, and twelve horizontal gene transfer (HGT) fragments were found in C. tianeensis, Camellia huana, Camellia liberofilamenta, and C. nitidissima, respectively. The phylogenetic tree clusters the four species of sect. Chrysantha plants into one group, and C. huana and C. liberofilamenta have closer affinities.ConclusionsIn this study, the mitochondrial genomes of four sect. Chrysantha plants were assembled and annotated, and these results contribute to the development of new genetic markers, DNA barcode databases, genetic improvement and breeding, and provide important references for scientific research, population genetics, and kinship identification of sect. Chrysantha plants.

Isolation and purification of DNA double-strand break repair intermediates for understanding complex molecular mechanisms.

Branched DNA molecules are key intermediates in the molecular pathways of DNA replication, repair and recombination. Understanding their structural details, therefore, helps to envisage the mechanisms underlying these processes. While the configurations of DNA molecules can be effectively analysed in bulk using gel electrophoresis techniques, direct visualization provides a complementary single-molecule approach to investigating branched DNA structures. However, for microscopic examination, the sample needs to be free from impurities that could obscure the molecules of interest, and free from the bulk of unwanted non-specific DNA molecules that would otherwise dominate the field of view. Additionally, in the case of recombination intermediates, the length of the DNA molecules becomes an important factor to consider since the structures can be spread over a large distance on the chromosome in vivo. As a result, apart from sample purity, efficient isolation of large-sized DNA fragments without damaging their branched structures is crucial for further analysis. These factors are illustrated by the example of DNA double-strand break repair in the bacterium E. coli. In E. coli recombination intermediates may be spread over a distance of 40 kb which constitutes less than 1% of the 4.6 Mb genome. This study reveals ways to overcome some of the technical challenges that are associated with the isolation and purification of large and complex branched DNA structures using E. coli DNA double-strand break repair intermediates. High-molecular weight and branched DNA molecules do not run into agarose gels subjected to electrophoresis. However, they can be extracted from the wells of the gels if they are agarose embedded, by using β-agarase digestion, filtration, and concentration. Furthermore, a second round of gel electrophoresis followed by purification is recommended to enhance the purity of the specific DNA samples. These preliminary findings may prove to be pioneering for various single-molecule analyses of large and complex DNA molecules of DNA replication, repair and recombination.

Highly sensitive and specific electrochemical biosensor for direct detection of hepatitis C virus RNA in clinical samples using DNA strand displacement

Hepatitis C virus (HCV) is a common blood-borne infection that can lead to long-term illnesses such as hepatocellular cancer and liver cirrhosis. Early diagnosis is crucial for effective management, as no vaccine is available for preventing HCV infection. However, the high cost and complexity of current molecular diagnostic tools hinder efforts to achieve early diagnosis and prevent transmission, particularly in resource-limited settings. We developed a novel electrochemical biosensor for point-of-care testing (POCT) of HCV RNA. The sensor utilizes a strand displacement method, where the target RNA displaces a gold nanoparticle-labeled reporter probe (AuRP) from a pre-hybridized duplex with a magnetic nanoparticle (MNP)-labeled capture probe. The amount of displaced AuRP, detected using differential pulse anodic stripping voltammetry (DPASV), is directly proportional to the target RNA concentration. The biosensor exhibited excellent analytical performance, with a detection limit of 4 fM for synthetic targets and 43 ng/µL for RT-PCR products. Importantly, it successfully detected HCV RNA directly in clinical plasma samples without the need for RNA extraction or amplification. The sensor was used to analyze 30 RNA samples from HCV-positive patients, 20 cDNA samples from viral RNA, 30 HCV-positive plasma samples, and 22 HCV-negative plasma samples. The sensor results show good concordance with the RT-PCR results, demonstrating the sensor’s potential for detecting HCV in clinical samples.

Unwinding DNA strands by single-walled carbon nanotubes: Molecular docking and MD simulation approach

Despite the growing research into the use of carbon nano-tubes (CNTs) in science and medicine, concerns about their potential toxicity remain insufficiently studied. This study utilizes molecular docking calculations combined by molecular dynamics simulations to investigate the dynamic intricacies of the interaction between single-walled carbon nanotubes (swCNTs) and double-stranded DNA (dsDNA). By examining the influence of swCNT characteristics such as length, radius, and chirality, our findings shed light on the complex interplay that shapes the binding affinity and stability of the dsDNA-swCNT complex. Molecular docking results identify a zigzag swCNT, with a radius of 0.16 Å and a length of 38 Å, as exhibiting the highest binding affinity with dsDNA (−23.9 kcal/mol). Comprehensive analyses, spanning docking results, binding energies, RMSD, radius of gyration, and potential of mean force (PMF) profiles, provide a detailed understanding of the denaturation dynamics. The PMF profiles reveal the thermodynamic feasibility of the DNA-CNT interaction, outlining distinct energy landscapes and barriers: when the selected swCNT binds within the dsDNA groove, the system becomes trapped at the first and second local energy minima, occurring at 1.48 nm and 1.00 nm, respectively. Intramolecular hydrogen bond calculations show a significant reduction, affirming the denaturing effect of swCNTs on DNA. Furthermore, the study reveals a significant reduction in the binding affinity of Ethidium Bromide (EB) to dsDNA following its interaction with swCNT, with a decrease in EB binding to dsDNA of approximately 13.2 %. This research offers valuable insights into the toxic effects of swCNTs on dsDNA, contributing to a rationalization of the cancerous potential of swCNTs.

Geometry of Braided DNA Dictates Supercoiling Partition.

During DNA replication, the replisome must rotate relative to the DNA substrate, generating supercoiling that must be partitioned in front of or behind the replisome. Supercoiling partitioned behind the replisome may intertwine (or braid) daughter DNA molecules and restrict chromosome segregation. Supercoiling partitioning and torsional resistance at the replisome should depend on the geometry of the two daughter DNA molecules, determined by their end separations. However, experimental investigation of DNA braiding under well-defined DNA geometry has proven challenging. Here, we present methods to engineer braiding substrates of defined geometry, from minimal to significant end separations. We then directly measured the torque required to braid these substrates using an angular optical trap (AOT) and found that the torque required to initiate the braiding during the first 0.5 turn critically depends on the end separation. Once braiding started, we found that the subsequent effective twist persistence length of DNA braiding is about 20-30 nm, insensitive to the end separations. Our work highlights the crucial role of braiding geometry in dictating supercoiling partitioning and torque build-up during replication. It suggests that dynamic modulation of end separation on the daughter DNA molecules could serve as a mechanism to regulate replication progression in vivo .

A model for transcription-dependent R-loop formation at double-stranded DNA breaks: Implications for their detection and biological effects

R-loops are structures containing an RNA-DNA duplex and an unpaired DNA strand. During R-loop formation an RNA strand invades the DNA duplex, displacing the homologous DNA strand and binding the complementary DNA strand. Here we analyze a model for transcription-dependent R-loop formation at double-stranded DNA breaks (DSBs). In this model, R-loop formation is preceded by detachment of the non-template DNA strand from the RNA polymerase (RNAP). Then, strand exchange is initiated between the nascent RNA and the non-template DNA strand. During that strand exchange the length of the R-loop could either increase, or decrease in a biased random-walk fashion, in which the bias would depend upon the DNA sequence. Eventually, the restoration of the DNA duplex would completely displace the RNA. However, as long as the RNAP remains bound to the template DNA strand it prevents that displacement. Thus, according to the model, RNAPs stalled at DSBs can increase the lifespan of R-loops, increasing their detectability in experiments, and perhaps enhancing their biological effects.

Artificial Intelligence-Enhanced Analysis of Genomic DNA Visualized with Nanoparticle-Tagged Peptides under Electron Microscopy.

DNA visualization has advanced across multiple microscopy platforms, albeit with limited progress in the identification of novel staining agents for electron microscopy (EM), notwithstanding its ability to furnish a broad magnification range and high-resolution details for observing DNA molecules. Herein, a non-toxic, universal, and simple method is proposed that uses gold nanoparticle-tagged peptides to stain all types of naturally occurring DNA molecules, enabling their visualization under EM. This method enhances the current DNA visualization capabilities, allowing for sequence-specific, genomic-scale, and multi-conformational visualization. Importantly, an artificial intelligence (AI)-enabled pipeline for identifying DNA molecules imaged under EM is presented, followed by classification based on their size, shape, or conformation, and finally, extraction of their significant dimensional features, which to the best of authors' knowledge, has not been reported yet. This pipeline strongly improved the accuracy of obtaining crucial information such as the number and mean length of DNA molecules in a given EM image for linear DNA (salmon sperm DNA) and the circumferential length and diameter for circular DNA (M13 phage DNA), owing to its image segmentation capability. Furthermore, it remained robust to several variations in the raw EM images arising from handling during the DNA staining stage.

MethylSeqLogo: DNA methylation smart sequence logos

BackgroundSome transcription factors, MYC for example, bind sites of potentially methylated DNA. This may increase binding specificity as such sites are (1) highly under-represented in the genome, and (2) offer additional, tissue specific information in the form of hypo- or hyper-methylation. Fortunately, bisulfite sequencing data can be used to investigate this phenomenon.MethodWe developed MethylSeqLogo, an extension of sequence logos which includes new elements to indicate DNA methylation and under-represented dimers in each position of a set binding sites. Our method displays information from both DNA strands, and takes into account the sequence context (CpG or other) and genome region (promoter versus whole genome) appropriate to properly assess the expected background dimer frequency and level of methylation. MethylSeqLogo preserves sequence logo semantics—the relative height of nucleotides within a column represents their proportion in the binding sites, while the absolute height of each column represents information (relative entropy) and the height of all columns added together represents total informationResultsWe present figures illustrating the utility of using MethylSeqLogo to summarize data from several CpG binding transcription factors. The logos show that unmethylated CpG binding sites are a feature of transcription factors such as MYC and ZBTB33, while some other CpG binding transcription factors, such as CEBPB, appear methylation neutral.ConclusionsOur software enables users to explore bisulfite and ChIP sequencing data sets—and in the process obtain publication quality figures.

High fidelity DNA ligation prevents single base insertions in the yeast genome

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3–5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1Cdc9-dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome.

Genome-wide DNA methylation and their transgenerational pattern differ in Arabidopsis thaliana populations originated along the elevation of West Himalaya

Methylation at 5' cytosine of DNA molecule is an important epigenetic mark. It is known to play critical role in adaptation of organisms under different biotic and abiotic stressors via modulating gene expression and/or chromatin architecture. Plant populations evolved under variable climatic conditions may have evolved different epigenetic marks including DNA methylation. Here we, describe the genome-wide DNA methylation pattern under native field, F1 and F6 generation followed by their association with phenotypes, climate and global gene expression in the three Arabidopsis thaliana populations originated at different elevation ranges of Indian West Himalaya. We show that the global methyl cytosine (mC) content is more or less similar in the three populations but differ in their distribution across genome. There was an increase in differential methylation between the populations as elevation increased. The methylation divergence was the highest between the low and the high elevation populations. The high elevation populations were hypo-methylated than the low elevation population. The methylation in the genes was associated with population specific phenotypes and climate of the region. The genes which were differentially methylated as well as differentially expressed between the low and high elevation populations were mostly related to abiotic stresses. When grown under controlled condition, there was gain of differential methylation over native condition and the maximum percent changes was observed in CHH-sequence context. Further ~ 99.8% methylated cytosines were stably passed on from F1 to F6 generation. Overall, our data suggest that high elevation population is epigenetically more plastic under changing environmental condition.BackgroundArabidopsis thaliana is the model plant species and has been extensively studied to understand plants life processes. There are numerous reports on its origin, demography, evolution, epigenomes and adaptation etc. however, Indian populations of Arabidopsis thaliana evolved along wide elevation ranging from ~ 700 m amsl to ~ 3400 m amsl not explored yet. Here we, describe the genome-wide DNA methylation pattern under native field, F1 and F6 generation followed by their association with phenotypes, climate and global gene expression in the three Arabidopsis thaliana populations originated at different elevation ranges of Indian West Himalaya.Results In our study we found that total mCs percent was more or less similar in the three populations but differ in their distribution across genome. The proportion of CG-mCs was the highest, followed by CHH-mCs and CHG-mCs in all the three populations. Under native field condition the methylation divergence was more prominent between low and high elevation populations and the high elevation populations were hypo-methylated than the low elevation population. The methylation in the genes was linked to population-specific phenotypes and the regional climate. The genes that showed differential methylation and expression between low and high elevation populations were primarily associated with abiotic stress responses. When grown under controlled condition, there was gain of differential methylation compared to the native condition and the maximum percent changes was observed in CHH-sequence context. Further 99.8% methylated cytosines were stably passed on from F1 to F6 generation.Conclusions The populations of A. thaliana adapted at different climatic conditions were significantly differentially methylated both under native and controlled condition. However, the magnitude and extent of gain or loss of methylation were most significant between the low and the high elevation populations. Overall, our data suggest that high elevation population is epigenetically more plastic under changing environmental condition.

Complex transcriptional regulations of a hyperparasitic quadripartite system in giant viruses infecting protists

Hyperparasitism is a common pattern in nature that is not limited to cellular organisms. Giant viruses infecting protists can be hyperparasitized by smaller ones named virophages. In addition, both may carry episomal DNA molecules known as transpovirons in their particles. They all share transcriptional regulatory elements that dictate the expression of their genes within viral factories built by giant viruses in the host cytoplasm. This suggests the existence of interactions between their respective transcriptional networks. Here we investigated Acanthamoeba castellanii cells infected by a giant virus (megavirus chilensis), and coinfected with a virophage (zamilon vitis) and/or a transpoviron (megavirus vitis transpoviron). Infectious cycles were monitored through time-course RNA sequencing to decipher the transcriptional program of each partner and its impact on the gene expression of the others. We found highly diverse transcriptional responses. While the giant virus drastically reshaped the host cell transcriptome, the transpoviron had no effect on the gene expression of any of the players. In contrast, the virophage strongly modified the giant virus gene expression, albeit transiently, without altering the protein composition of mature viral particles. The virophage also induced the overexpression of transpoviron genes, likely through the indirect upregulation of giant virus-encoded transcription factors. Together, these analyses document the intricated transcriptionally regulated networks taking place in the infected cell.