Biology subjects

Singh, I.

Publications and source records attributed to Singh, I..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Intrinsic epigenetic state of primary osteosarcoma drives metastasis

Osteosarcoma (OS) is the most common primary malignant bone tumor affecting the pediatric population with high potential to metastasize to distal sites, most commonly the lung. Insights into defining molecular features contributing to metastatic potential are lacking. We have mapped the active chromatin landscapes of OS tumors by integrating histone H3 lysine acetylated chromatin (H3K27ac) profiles (n=13), chromatin accessibility profiles (n=11) and gene expression (n=13) to understand the differences in their active chromatin profiles and its impact on molecular mechanisms driving the malignant phenotypes. Primary OS tumors from patients with metastasis (primary met) have a distinct active chromatin landscape compared to primary tumors from patients without metastatic disease (localized). The difference in chromatin activity shapes the transcriptional profile of OS. We identified novel candidate genes involved in OS pathogenesis and metastasis, including PPP1R1B, PREX1 and IGF2BP1, which exhibit increased chromatin activity in primary met along with higher transcript levels. Overall, differential chromatin activity in primary met occurs in proximity of genes regulating actin cytoskeleton organization, cellular adhesion, and extracellular matrix suggestive of their role in facilitating OS metastasis. Furthermore, chromatin profiling of tumors from metastatic lung lesions noted increases in chromatin activity in genes involved in cell migration and key intracellular signaling cascades, including the Wnt pathway. Thus, this data demonstrates that metastatic potential is intrinsically present in primary metastatic tumors and the cellular chromatin profiles further adapt to allow for successful dissemination, migration, and colonization at the distal metastatic site.

cancer biology↗

bioRxiv2023

The human Y and inactive X chromosomes similarly modulate autosomal gene expression

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex-chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors - ZFX and ZFY - encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes.

genetics↗

bioRxiv2023

E2F1 Mediates SOX17 Deficiency-Induced Pulmonary Hypertension

RationaleRare genetic variants and genetic variation at loci in an enhancer in SRY-Box Transcription Factor 17 (SOX17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutation in SOX17 in PAH pathogenesis has not been reported. ObjectivesTo investigate the role of SOX17 deficiency in pulmonary hypertension (PH) development. MethodsHuman lung tissue and endothelial cells (ECs) from IPAH patients were used to determine the expression of SOX17. Tie2Cre-mediated and EC-specific deletion of Sox17 mice were assessed for PH development. Single-cell RNA sequencing analysis, human lung ECs, and smooth muscle cell culture were performed to determine the role and mechanisms of SOX17 deficiency. A pharmacological approach was used in Sox17 deficiency mice for therapeutic implication. Measurement and Main ResultsSOX17 expression was downregulated in the lungs and pulmonary ECs of IPAH patients. Mice with Tie2Cre mediated Sox17 knockdown and EC-specific Sox17 deletion developed spontaneously mild PH. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced PH in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and anti-apoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP signaling. E2F Transcription Factor 1 (E2F1) signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction and PH development. ConclusionsOur study demonstrated that endothelial SOX17 deficiency induces PH through E2F1 and targeting E2F1 signaling represents a promising approach in PAH patients.

physiology↗

bioRxiv2023

Structure-based discovery of inhibitors of the SARS-CoV-2 Nsp14 N7-methyltransferase

An under-explored target for SARS-CoV-2 is non-structural protein 14 (Nsp14), a crucial enzyme for viral replication that catalyzes the methylation of N7-guanosine of the viral RNA at 5'-end; this enables the virus to evade the host immune response by mimicking the eukaryotic post-transcriptional modification mechanism. We sought new inhibitors of the S-adenosyl methionine (SAM)-dependent methyltransferase (MTase) activity of Nsp14 with three large library docking strategies. First, up to 1.1 billion make-on-demand ("tangible") lead-like molecules were docked against the enzymes SAM site, seeking reversible inhibitors. On de novo synthesis and testing, three inhibitors emerged with IC50 values ranging from 6 to 43 M, each with novel chemotypes. Structure-guided optimization and in vitro characterization supported their non-covalent mechanism. In a second strategy, docking a library of 16 million tangible fragments revealed nine new inhibitors with IC50 values ranging from 12 to 341 M and ligand efficiencies from 0.29 to 0.42. In a third strategy, a newly created library of 25 million tangible, virtual electrophiles were docked to covalently modify Cys387 in the SAM binding site. Seven inhibitors emerged with IC50 values ranging from 3.2 to 39 M, the most potent being a reversible aldehyde. Initial optimization of a second series yielded a 7 M acrylamide inhibitor. Three inhibitors characteristic of the new series were tested for selectivity against 30 human protein and RNA MTases, with one showing partial selectivity and one showing high selectivity. Overall, 32 inhibitors encompassing eleven chemotypes had IC50 values <50 M and 5 inhibitors in four chemotypes had IC50 values <10 M. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing multiple starting points for optimizing towards antiviral activity.

biophysics↗

bioRxiv2022

Regenerative Agriculture Augments Bacterial Community Structure for a Healthier Soil and Agriculture

Use of chemical fertilization and pesticides not only harm the environment but also have detrimental consequences on human health. In recent years, there has been a major emphasis worldwide on natural agriculture methods. Regenerative agriculture is known across the world as a combination of nature-friendly farming practices such as no-till, cover cropping, crop-rotation, agro-forestry and use of organic home-based/farm-based ingredients to revive soil health. In India, a number of farmers are slowly adopting these practices using home-based mixtures and farmyard manure for soil rejuvenation and pest management. In order to evaluate the efficacy of the regenerative agriculture practices, this study compared conventional and regenerative agriculture plots for their soil bacterial and nutrient profiles. Two crops - ragi and vegetable (tomato/beans), and different lengths ([≤]3 and >5 years) of regenerative practices were additional metrics considered to understand variabilities due to crop-type and period of application. We found that all regenerative practices were effective in bringing about an enrichment for soil bacteria with a more heterogeneous composition. Additionally, the regenerative vegetable (RV) plots had an enhanced representation of Actinobacteriota, Chloroflexi, Cyanobacteria and Patescibacteria in comparison to conventional vegetable (CV) plots and Barren land (BL). Similarly, the regenerative ragi (RR) plots saw higher representation of Firmicutes and Actinobacteriota in comparison to conventional ragi (CR) plots and BL. The RV plots were also found to be enriched for Plant Growth Promoting Rhizobacteria (PGPRs) - Pseudomonas sp., and RR plots were enriched for Bacillus sp., and Mesorhizobium sp., which are known to play significant roles in vegetable and ragi growth respectively. Interestingly, long-term regenerative agriculture was able to support good nutrient composition while enhancing Soil Organic Carbon (SOC) levels. In all, the regenerative agriculture practices were found to be effective in improving bacterial community structure and simultaneously improving soil health. We found that BL soil with eucalyptus plantation showed least bacterial diversity suggesting detrimental impact on soil health.

ecology↗

bioRxiv2022

The time to initiate retrieval of a memory depends on recency

Continuous recognition has long been used to study the recency effect in recognition memory. In continuous recognition, response time (RT) increases as a logarithmic function of the lag since a probe was last presented. Although this could simply be due to decaying trace strength, careful examination of response time (RT) distributions showed that the onset of RT distributions changed with the logarithm of the lag since a probe was originally presented. Each doubling of lag resulted in a shift of roughly 20 ms in the rise time of the RT distributions. To test the hypothesis that this increase was simply due to increased facility in processing the probe item, Experiment six repeated items six times. Repetition resulted in faster RTs, but did not change the effect of log lag on RT. In light of recent neurophysio-logical evidence, we consider the hypothesis that memory requires a recovery of temporal context, and the time to retrieve a prior temporal context goes up with the logarithm of the time since it was experienced.

animal behavior and cognition↗

bioRxiv2022

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors

Antiviral therapeutics to treat SARS-CoV-2 are much desired for the on-going pandemic. A well-precedented viral enzyme is the main protease (MPro), which is now targeted by an approved drug and by several investigational drugs. With the inevitable liabilities of these new drugs, and facing viral resistance, there remains a call for new chemical scaffolds against MPro. We virtually docked 1.2 billion non-covalent and a new library of 6.5 million electrophilic molecules against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 M and 20 M, respectively. Several series were optimized, resulting in inhibitors active in the low micromolar range. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. Together, these compounds reveal new chemotypes to aid in further discovery of MPro inhibitors for SARS-CoV-2 and other future coronaviruses.

biophysics↗

bioRxiv2022

Structure-based Discovery of Conformationally Selective Inhibitors of the Serotonin Transporter

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has an unusual anti-depressant profile and stabilizes the inward-open conformation. Unfortunately, ibogaine is promiscuous and cardiotoxic, limiting understanding of inward-open state ligands. We computationally docked over 200 million small molecules against the ibogaine stabilized inward-open state of SERT. Thirty-six top-ranking compounds were synthesized and thirteen inhibited with potencies ranging from 29 to 5000 nM. Structure-based optimization led to two novel inhibitors with Ki values down to 3 nM. The new molecules stabilized an outward-closed state of the transporter and had little activity against off-targets. A cryo-EM structure of one of these bound to SERT confirmed the predicted geometry. In mouse behavioral assays, both had anxiolytic and anti-depressant activity, with potencies up to 200 better than fluoxetine.

biochemistry↗

bioRxiv2022

Autoantibody and hormone activation of the thyrotropin G protein-coupled receptor

Thyroid hormones are vital to growth and metabolism. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR). Autoantibodies that activate the TSHR pathologically increase thyroid hormones in Graves disease. How autoantibodies mimic TSH function remains unclear. We determined cryogenic-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. TSH selects an upright conformation of the extracellular domain due to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody selects a similar upright conformation of the extracellular domain. Conformational changes in the extracellular domain are transduced to the seven transmembrane domain via a conserved hinge domain, a tethered peptide agonist, and a phospholipid that binds within the seven transmembrane domain. Rotation of the TSHR ECD relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to G protein-coupled receptors with large extracellular domains.

biochemistry↗

bioRxiv2021

Analyses of the Autism-associated Neuroligin-3 R451C Mutation in Human Neurons Reveals a Gain-of-Function Synaptic Mechanism

Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C-mutation on human neurons has not been investigated. Here, we generated human knock-in neurons with the NLGN3 R451C-mutation. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C-mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses. No significant cell death and endoplasmic reticulum stress were detected. Importantly, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons that corresponded to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C-mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASDs.

neuroscience↗

bioRxiv2021

Energy Penalties Enhance Flexible Receptor Docking in a Model Cavity

Protein flexibility remains a major challenge in library docking due to difficulties in sampling conformational ensembles with accurate probabilities. Here we use the model cavity site of T4 Lysozyme L99A to test flexible receptor docking with energy penalties from molecular dynamics (MD) simulations. Crystallography with larger and smaller ligands indicates that this cavity can adopt three major conformations, open, intermediate, and closed. Since smaller ligands typically bind better to the cavity site, we anticipate an energy penalty for cavity opening. To estimate its magnitude, we calculate conformational preferences from MD simulations. We find that including a penalty term is essential for retrospective ligand enrichment, otherwise high-energy states dominate the docking. We then prospectively docked a library of over 900,000 compounds for new molecules binding to each conformational state. Absent a penalty term, the open conformation dominated the docking results; inclusion of this term led to a balanced sampling of ligands against each state. High ranked molecules were experimentally tested by Tm-upshift and X-ray crystallography. From 33 selected molecules, we identified 18 new ligands and determined 13 crystal structures. Most interesting were those bound to the open cavity, where the buried site opens to bulk solvent. Here, highly unusual ligands for this cavity had been predicted, including large ligands with polar tails; these were confirmed both by binding and by crystallography. In docking, incorporating protein flexibility with thermodynamic weightings may thus access new ligand chemotypes. The MD approach to accessing and, crucially, weighting such alternative states may find general applicability. Significance StatementThe dynamic nature of biomolecules is typically neglected in docking screens for ligand discovery. Key to benefitting from various receptor conformations is not only structural but also thermodynamic information. Here we test a general approach that uses conformational preferences from enhanced and conventional MD simulations to account for the cost of transitions to high energy states. Including this information as a conformational penalty term in a docking scoring function, we perform retrospective and prospective screens and experimentally confirm novel ligands with Tm-upshift and X-ray crystallography.

biophysics↗

bioRxiv2021

Fine-tuning the transcriptional regulatory model of adaptation response to phosphate stress in maize (Zea mays L.)

The post green revolution agriculture is based on generous application of fertilizers and high-yielding genotypes that are suited for such high input regimes. Cereals, like maize (Zea mays L.) are capable of utilizing less than 20% of the applied inorganic phosphate (Pi) - a non-renewable fertilizer resource. A greater understanding of the molecular mechanisms underlying the acquisition, transportation and utilization of Pi may lead to strategies to enhance phosphorus use efficiency (PUE) in field crops. In this study, we selected 12 Pi responsive genes in maize and carried out their comparative transcriptional expression in root and leaf tissues of a hydroponically grown Pi stress tolerant maize inbred line HKI-163, under sufficient and deficient Pi conditions. Pi starvation led to significant increase in root length; marked proliferation of root hairs and lesser number of crown roots. Eleven genes were significantly up or down regulated in Pi deficient condition. The putative acid phosphatase, ZmACP5, expression was up regulated by 162.81 and 74.40 fold in root and leaf tissues, respectively. The RNase, ZmRNS1 showed 115 fold up regulation in roots under Pi deprivation. Among the two putative high affinity Pi transporters ZmPht1;4 was found specific to root, whereas ZmPht2 was found to be up regulated in both root and leaf tissues. The genes involved in Pi homeostasis pathway (ZmSIZ1, SPX1 and Pho2) were up regulated in root and leaf. In light of the expression profiling of selected regulatory genes, an updated model of transcriptional regulation under Pi starvation in maize has been proposed.

plant biology↗

bioRxiv2021

Pharmacological Inhibition of BTK reduces neuroinflammation and stress induced anxiety in vivo

Stress related disorders lead to serious psychiatric disabilities and are comorbid with anxiety and depression. Current therapies targeting several neurotransmitter systems are only able to mitigate symptoms partially. It is well recognized that stress and trauma related disorders lead to a prominent inflammatory response in humans, and in several animal models a robust neuroinflammatory response has been observed. However, the therapeutic potential of targeting specific components of the inflammatory response has not been adequately studied in this context. The current study investigated the NLRP3 - Caspase1-IL-1{beta} pathway, which recent research has identified as a major contributor to exacerbated inflammatory response in several peripheral and central nervous system pathological conditions. Using two different models of stress, first - single prolonged restraint stress followed by brief underwater submersion and second - predator odor exposure in mice, we demonstrate heightened anxious behavior in mice one-week after stress. Females in both models display an exacerbated anxiety response than males within the stressed group. Consistent with this data stressed animals demonstrate upregulation of IL-1{beta}, IL-6, Caspase1 activity and NLRP3 inflammasome activation in brain, with female animals showing a stronger neuroinflammatory phenotype. Pharmacological inhibition of NLRP3 inflammasome activation led to a rescue in terms of anxious behavior as well as attenuated neuroinflammatory response, both of which were significantly more prominent in female animals. Further, we observed induction of activated Brutons Tyrosine Kinase (BTK), an upstream positive regulator of NLRP3 inflammasome activation, in hippocampus and amygdala of stressed mice. Next, we conducted proof-of-concept pharmacological BTK inhibitor studies with Ibrutinib, a drug that is already FDA approved for use in certain types of lymphomas and leukemias, as well as a second inhibitor of BTK, LFM-A13. In both sets of experiments, we found inhibition of BTK significantly reduced the anxious behavior in stressed mice and attenuated the induction of NLRP3 inflammasome, Caspase 1 and IL1{beta}. Our results suggest that BTK inhibition can be further investigated in context of human stress and trauma related disorders as a therapeutic strategy.

neuroscience↗

bioRxiv2020

Human PRE-PIK3C2B exhibits long-range intra- and inter-chromosomal interactions with genomic regions enriched in repressive marks.

Human PRE-PIK3C2B is a dual nature polycomb response element that interacts with both polycomb as well as trithorax members. In the current study, using 4C-Seq (Capturing Circular Chromosomal Conformation-Sequencing), we identified long-range chromatin interactions associated with PRE-PIK3C2B and validated them with 3C-PCR. We identified both intra-as well as inter-chromosomal interactions, a large proportion of which were found to be closely distributed around transcriptional start sites (TSS). A significant number of interactions were also found to be associated with heterochromatic regions. Meta-analysis of ENCODE ChIP-Seq data identified an overall enrichment of YY1, CTCF as well as histone modification such as H3K4me3 and H3K27me marks in different cell lines. Almost 90% interactions were derived from either intronic or intergenic regions. among which large proportions of intronic interactors were either unique sequences or LINE/SINE derived. In case of intergenic interactions, majority of the interaction were associated with LINE/SINE repeats. We further found that genes proximal to the interactor sequences were co-expressed, they showed reduced expression. To the best of our knowledge this is one of the early demonstrations of long-range interaction of PRE sequences in human genome.

genetics↗

bioRxiv2020

PRAK-03202: A triple antigen VLP vaccine candidate against SARS CoV-2

The rapid development of safe and effective vaccines against SARS CoV-2 is the need of the hour for the coronavirus outbreak. Here, we have developed PRAK-03202, the worlds first triple antigen VLP vaccine candidate in a highly characterized S. cerevisiae-based D-CryptTM platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunizations using three different doses of PRAK-03202 induces antigen specific (Spike, envelope and membrane proteins) humoral response and neutralizing potential. PBMCs from convalescent patients, when exposed to PRAK-03202, showed lymphocyte proliferation and elevated IFN-{gamma} levels suggestive of conservation of epitopes and induction of T helper 1 (Th1)-biased cellular immune responses. These data support the clinical development and testing of PRAK-03202 for use in humans.

immunology↗

bioRxiv2020

Multi-Antigenic Virus-like Particle of SARS CoV-2 produced in Saccharomyces cerevisiae as a vaccine candidate

Spike, Envelope and Membrane proteins from the SARS CoV-2 virus surface coat are important vaccine targets. We hereby report recombinant co-expression of the three proteins (Spike, Envelope and Membrane) in a engineered Saccharomyces cerevisiae platform (D-Crypt) and their self-assembly as Virus-like particle (VLP). This design as a multi-antigenic VLP for SARS CoV-2 has the potential to be a scalable vaccine candidate. The VLP is confirmed by transmission electron microscopy (TEM) images of the SARS CoV-2, along with supportive HPLC, Dynamic Light Scattering (DLS) and allied analytical data. The images clearly outline the presence of a "Corona" like morphology, and uniform size distribution.

biochemistry↗

bioRxiv2020

ACE2 Homo-dimerization, Human Genomic variants and Interaction of Host Proteins Explain High Population Specific Differences in Outcomes of COVID19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive single-stranded RNA virus that causes a highly contagious Corona Virus Disease (COVID19). Entry of SARS-CoV-2 in human cells depends on binding of the viral spike (S) proteins to cellular receptor Angiotensin-converting enzyme 2 (ACE2) and on S-protein priming by host cell serine protease TMPRSS2. Recently, COVID19 has been declared pandemic by World Health Organization (WHO) yet high differences in disease outcomes across countries have been seen. We provide evidences to explain these population-level differences. One of the key factors of entry of the virus in host cells presumably is because of differential interaction of viral proteins with host cell proteins due to different genetic backgrounds. Based on our findings, we conclude that a higher expression of ACE2 is facilitated by natural variations, acting as Expression quantitative trait loci (eQTLs), with different frequencies in different populations. We suggest that high expression of ACE2 results in homo-dimerization, proving disadvantageous for TMPRSS2 mediated cleavage of ACE2; whereas, the monomeric ACE2 has higher preferential binding with SARS-CoV-2 S-Protein vis-a-vis its dimerized counterpart. Further, eQTLs in TMPRSS2 and natural structural variations in the gene may also result in differential outcomes towards priming of viral S-protein, a critical step for entry of the Virus in host cells. In addition, we suggest that several key host genes, like SLC6A19, ADAM17, RPS6, HNRNPA1, SUMO1, NACA, BTF3 and some other proteases as Cathepsins, might have a critical role. To conclude, understanding population specific differences in these genes may help in developing appropriate management strategies for COVID19 with better therapeutic interventions.

genetics↗

bioRxiv2020

Positioning of nucleosomes containing γ-H2AXprecedes active DNA demethylation and transcription initiation

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of HMG proteins during transcription initiation remain unclear. Here we show that HMG AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT (facilitates chromatin transcription) complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 ({gamma}-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure the integrity of the genome.

molecular biology↗