Chromosomal DNA Research Articles

IRF-1 Regulates Mitochondrial Respiration and Intrinsic Apoptosis Under Metabolic Stress through ATP Synthase Ancillary Factor TMEM70.

Mitochondrial dysfunction, which can be caused by metabolic stressors such as oxidized low-density lipoprotein (oxLDL), sensitizes the endothelium to pathological changes. The transcription factor interferonregulatoryfactor 1 (IRF-1) is a master regulator of inflammation, previously shown to promote oxLDL-induced inflammatory pyroptosis in human aortic endothelial cells (HAEC). However, a presumed role for IRF-1 in regulating the intrinsic apoptotic pathway in response to metabolic stress has not been demonstrated. Here targeted deletion of IRF-1 by siRNA in HAEC aggravated oxLDL-induced, mitochondria-mediatedintrinsicapoptosis, as evidenced by increased Caspase-3 and Caspase-9 activation, and chromosomal DNA breakage. The increased apoptosis was concomitant with accumulation of mitochondrial ROS, decrease in intracellular ATP production and respiratory oxygen consumption, and abnormal mitochondrial structure. RNA profiling of endothelial cells isolated from wild type and Irf1 knockout mice, followed by quantitative PCR, luciferase activity assay and chromatin immunoprecipitation (ChIP), revealed that IRF-1 directly regulated the expression of transmembrane protein 70 (TMEM70), an ancillary factor required for the assembly of ATP synthase and conversion of an electrochemical gradient to ATP synthesis. Mirroring the effect of IRF1 knockdown, depletion of TMEM70 in HAEC resulted in impaired mitochondrial function and enhanced cell apoptosis. In contrast, overexpression of TMEM70 rescued ATP biosynthesis and suppressed apoptosis in oxLDL-treated, IRF-1-deficient HAEC. These results reveal a novel homeostatic role for IRF-1 in the regulation of mitochondrial function and associated stress-induced apoptosis.

ALIGNMENT OF PREP USE WITH POTENTIAL HIV EXPOSURE IN YOUNG WOMEN AND MEN IN UGANDA.

Despite high oral pre-exposure prophylaxis (PrEP) uptake among young heterosexual cisgender women, early discontinuation is frequent. It is unclear whether this aligns with potential HIV exposure. Young women 16-25 years and ≥1 of their male partners were enrolled in separate but linked longitudinal HIV PrEP studies in Kampala, Uganda from 2018-2021. Data on sexual behavior, PrEP use, STI positivity, and Y chromosome DNA (Yc DNA; a marker for condomless sex) were collected at enrollment and quarterly visits. Potential HIV exposure was defined as one of the following in the past 3 months: any STI, detection of Yc DNA, condomless vaginal sex, or multiple sex partners. Alignment between potential HIV exposure and PrEP use by participants was examined using GEE regression. 88 young women (median age=20.6, IQR 19.5-22.0) and 124 male partners (median age=23.5, IQR 21.0-26.0) were included. Women and men were dispensed PrEP in 66.9% and 60.5% of their first linked visits, respectively. PrEP dispensation was more common when women or men self-reported condomless vaginal sex and multiple sex partners, or when women had Yc DNA detected in vaginal swabs. Men's self-report of multiple partners (aPR=1.56, p=0.012) and the detection of Yc DNA (aPR=1.52, p=0.040) were significantly associated with women's PrEP dispensation. Women and their male partners may align their PrEP use with their HIV risk behaviors, providing some reassurance that PrEP discontinuation in young people often aligns with sexual behavior. Greater attention to measurement of and mismatches in PrEP discontinuation and potential HIV exposure is needed.

Diagnostic status and new explorations in upper urinary tract urothelial carcinoma: a literature review.

Upper urinary tract urothelial carcinomas (UTUCs) are characterized by their obscure location, insidious onset, and propensity for early muscle-invasive and lymph node metastasis, resulting in a generally poor prognosis. Clinically, diagnosis primarily relies on urine cytology, computed tomography urography (CTU), and white light ureteroscopy (WL-URS). These methods, however, suffer from low sensitivity, high cost, inefficient testing, patient discomfort, surgery-related complications, and potential tumor implantation. This literature review summarizes the advantages and disadvantages of the existing diagnostic tools for UTUC and discusses promising advancements in the field. A comprehensive computer-assisted search, supplemented by literature tracing, was conducted to review publications on UTUCs diagnostics from the PubMed, Embase, and Web of Science databases covering the period from 2000 to August 2024. The research focused on synthesizing findings from the selected publications. Over 100 studies were reviewed, spanning from traditional diagnostic methods like urine cytology, CTU, and WL-URS, as well as newer methods including endoscopic advances, biopsy methods, radiomics, immunocytology [urinary tumor cell (UTC) assay], urinary genetic tests (identifying chromosomal abnormalities, DNA mutations, DNA methylation, and RNA), and hematological and urinary tests for microRNAs (miRNAs) and proteins (plasma protein classifiers). While traditional methods have been extensively evaluated, they remain suboptimal in terms of effectiveness, safety, patient satisfaction, and cost. Emerging diagnostics and molecular markers show promise, such as the high sensitivity of the urinary UTC assay for early-stage tumors and the non-invasive nature of various urinary molecular tests, along with the high accuracy of plasma protein classifiers. However, these new tools generally lack thorough evaluation and are often restricted to reports from single institutions or simple differentiation of cancerous cases from controls under experimental conditions. Traditional diagnostic methods for UTUCs exhibit significant limitations, which newer tools and molecular markers are poised to address. However, comprehensive evaluations of these innovations are required to confirm their efficacy.

ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse.

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.

The concept of natural genome reconstruction. Part 1. Basic provisions of the “natural genome reconstruction” concept. Changing the genome of hematopoietic stem cells using several natural cellular mechanisms that are inherent in the hematopoietic cell and determine its biological status as “the source of the body’s reparative potential”

We present a series of articles proving the existence of a previously unknown mechanism of interaction between hematopoietic stem cells and extracellular double-stranded DNA (and, in particular, double-stranded DNA of the peripheral bloodstream), which explains the possibility of emergence and fixation of genetic information contained in double-stranded DNA of extracellular origin in hematopoietic stem cells. The concept of the possibility of stochastic or targeted changes in the genome of hematopoietic stem cells is formulated based on the discovery of new, previously unknown biological properties of poorly differentiated hematopoietic precursors. The main provisions of the concept are as follows. The hematopoietic stem cell takes up and internalizes fragments of extracellular double-stranded DNA via a natural mechanism. Specific groups of glycocalyx factors, including glycoproteins/proteoglycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors, take part in the internalization event. The binding sites for DNA fragments are heparin-binding domains and clusters of positively charged amino acid residues that are parts of protein molecules of these factors. Extracellular fragments delivered to the internal compartments of hematopoietic stem cells initiate terminal differentiation, colony formation, and proliferation of hematopoietic precursors. The molecular manifestation of these processes is the emergence and repair of pangenomic single-strand breaks. The occurrence of pangenomic single-strand breaks and restoration of genome (genomic DNA) integrity are associated with activation of a “recombinogenic situation” in the cell; during its active phase, stochastic homologous recombination or other recombination events between extracellular fragments localized in the nucleus and chromosomal DNA are possible. As a result, genetic material of initially extracellular localization either integrates into the recipient genome with the replacement of homologous chromosomal segments, or is transitively present in the nucleus and can manifest itself as a new genetic trait. It is assumed that as a result of stochastic acts of homologous exchange, chromosome loci are corrected in hematopoietic stem cells that have acquired mutations during the existence of the organism, which are the cause of clonal hematopoiesis associated with old age. In this regard, there is a fundamental possibility of changing the hematopoietic status of hematopoietic stem cells in the direction of polyclonality and the original diversity of clones. Such events can form the basis for the rejuvenation of the blood-forming cell system. The results of the laboratory’s work indicate that other stem cells in the body capture extracellular DNA fragments too. This fact creates a paradigm for the overall rejuvenation of the body.

A Eukaryote-Featured Membrane Phospholipid Enhances Bacterial Formaldehyde Tolerance and Assimilation of One-Carbon Feedstocks.

Efficient bioassimilation of one-carbon (C1) feedstocks is often hindered by the toxicity of C1 substrates and/or intermediates. We compared the toxicity of several common C1 substrates/intermediates and found that formaldehyde imposes the highest toxicity on the representative bacterium Escherichia coli. Besides causing chromosomal DNA and protein damage effects, here, we revealed that formaldehyde greatly impairs cell membranes. To this end, here, we sought to remodel the cell membrane of E. coli by introducing a non-native, eukaryote-featured membrane phospholipid composition, phosphatidylcholine (PC). This engineered E. coli strain exhibited significantly increased membrane integrity, resulting in enhanced formaldehyde tolerance. When applied to C1 assimilation, the PC-harboring E. coli consumed up to 4.7 g/L methanol, which is 23-fold higher than that of the control strain (0.2 g/L). In summary, the present study highlights the detrimental impact of formaldehyde-induced membrane damage and thus underscores the significance of membrane remodeling in enhancing formaldehyde tolerance and facilitating the assimilation of C1 substrates.

EPCO-52. INVESTIGATING EGFR CONTAINING EXTRACHROMOSOMAL DNA DYNAMICS IN GBM

Abstract The epidermal growth factor receptor (EGFR) is the most frequently altered gene in GBM, with EGFR amplification occurring in approximately 40% of all glioblastomas, half of which exhibit the EGFRvIII deletion variant. EGFR amplification often occurs through the formation of extrachromosomal DNA (ecDNA), where EGFR typically co-amplifies with upstream enhancers. ecDNA is commonly found in a wide range of cancer types, particularly Glioblastoma (GBM), but is rare in normal cells. The uneven segregation of ecDNAs results in oncogene copy number variations in subclones, producing highly heterogeneous tumor cells and providing a selective advantage to cancer cells in response to changing environmental conditions. Meanwhile, ecDNAs keep evolving through fusion, deletion, or integration of new fragments, resulting in alterations to their structure and sequence. Some ecDNAs can reintegrate into chromosomal DNA, disrupting the integrity of chromosomal genes or rewiring chromosomal gene expression via ecDNA resident enhancers. However, the dynamics of EGFR-containing ecDNA (ecEGFR) have not been thoroughly investigated across primary and recurrent GBM. In this study, we profiled 47 glioblastoma specimens (both primary and recurrent) using a multi-omic approach, including WGS, RNA-seq, and ATAC-seq, identifying ecDNA in 14 samples, 10 of which carried EGFR. EGFR deletions were observed in 4 ecEGFR-positive samples, with 2 being the vIII variant. Interestingly, EGFR deletions predominantly occurred in the primary GBM. These ecEGFR samples exhibited significantly high EGFR copy number, gene expression, and chromatin accessibility. Additionally, there were samples with ecEGFR in the primary tumor but not in the recurrent tumor, or ecEGFR appears only in recurrent tumor. One patient showed an ecEGFR deletion in the primary tumor, while the recurrent tumor exhibited wild type ecEGFR.

Characterization of the enzyme for 5-hydroxymethyluridine production and its role in silencing transposable elements in dinoflagellates

Dinoflagellate chromosomes are extraordinary, as their organization is independent of architectural nucleosomes unlike typical eukaryotes and shows a cholesteric liquid crystal state. 5-hydroxymethyluridine (5hmU) is present at unusually high levels and its function remains an enigma in dinoflagellates chromosomal DNA for several decades. Here, we demonstrate that 5hmU contents vary among different dinoflagellates and are generated through thymidine hydroxylation. Importantly, we identified the enzyme, which is a putative dinoflagellate TET/JBP homolog, catalyzing 5hmU production using both in vivo and in vitro biochemical assays. Based on the near-chromosomal level genome assembly of dinoflagellate Amphidinium carterae, we depicted a comprehensive 5hmU landscape and found that 5hmU loci are significantly enriched in repeat elements. Moreover, inhibition of 5hmU via dioxygenase inhibitor leads to transcriptional activation of 5hmU-marked transposable elements, implying that 5hmU appears to serve as an epigenetic mark for silencing transposon. Together, our results revealed the biogenesis, genome-wide landscape, and molecular function of dinoflagellate 5hmU, providing mechanistic insight into the function of this enigmatic DNA mark.

The evolutionary landscape of prokaryotic chromosome/plasmid balance

The balance between chromosomal and plasmid DNAs determines the genomic plasticity of prokaryotes. Natural selections, acting on the level of organisms or plasmids, shape the abundances of plasmid DNAs in prokaryotic genomes. Despite the importance of plasmids in health and engineering, there have been rare systematic attempts to quantitatively model and predict the determinants underlying the strength of different selection forces. Here, we develop a metabolic flux model that describes the intracellular resource competition between chromosomal and plasmid-encoded reactions. By coarse graining, this model predicts a landscape of natural selections on chromosome/plasmid balance, which is featured by the tradeoff between phenotypic and non-phenotypic selection pressures. This landscape is further validated by the observed pattern of plasmid distributions in the vast collection of prokaryotic genomes retrieved from the NCBI database. Our results establish a universal paradigm to understand the prokaryotic chromosome/plasmid interplay and provide insights into the evolutionary origin of plasmid diversity.

DciA, the Bacterial Replicative Helicase Loader, promotes LLPS in the presence of ssDNA

The loading of the bacterial replicative helicase DnaB is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that DciA from Vibrio cholerae is composed of a globular domain followed by an unfolded extension and demonstrated its strong affinity for DNA. Here, we characterize the condensates formed by VcDciA upon interaction with a short single-stranded DNA substrate. We demonstrate the fluidity of these condensates using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of DnaB in the droplets, concomitant with the release of DciA. We show that the well-known helicase loader DnaC from Escherichia coli is also competent to form these phase-separated condensates in the presence of ssDNA. Our phenomenological data are still preliminary as regards the existence of these condensates in vivo, but open the way for exploring the potential involvement of DciA in the formation of non-membrane compartments within the bacterium to facilitate the assembly of replication players on chromosomal DNA.

A novel regulatory motif at the hinge dimer interface of the MksB mediates dimerization and DNA binding activity

The Structural Maintenance of Chromosome (SMC) protein is essential for various cellular processes, including chromosome organization, DNA repair, and genome stability. MksB, an alternative SMC protein present in prokaryotes, comprises a hinge dimerization domain and an ABC ATPase head domain linked by a coiled-coil arm. While hinge dimerization in bacterial and eukaryotic SMCs is attributed to conserved glycines, our study unveils the critical role of a novel KDDR motif located at a loop near the hinge dimer interface in Mycobacterium smegmatis MksB (MsMksB). We demonstrate the regulatory role of this motif in MsMksB dimerization and DNA binding activity. The K600D mutation in the KDDR motif induces MsMksB dimer-to-monomer conversion, highlighting the significance of this motif in MsMksB dimerization. Mass spectrometry-based mapping of the DNA binding site revealed the lysine's involvement in protein-DNA interaction. Monomers of the hinge domain lose DNA binding activity, and MsMksB single-arm mutants exhibit reduced DNA binding and ATPase activity, underscoring the importance of hinge-mediated dimerization in MsMksB function. Notably, the R603D mutant retains dimerization but shows compromised ATPase and DNA binding activities. Mutants with defective ATPase activity exhibit impaired DNA condensation in vivo. These findings provide novel regulatory insight into the mechanism of MksB dimerization and DNA binding, uncovering the fundamental processes of chromosome condensation and segregation.

Beneath the Surface: Neoantigens beyond Chromosomal DNA Mutations.

The conventional wisdom is that the overwhelming majority of neoantigens arise from chromosomal DNA mutations; however, recent studies show that posttranscriptional and posttranslational events can also generate neoantigens. This commentary provides an overview of known and potential sources of nonchromosomal neoantigens, emerging technologies, and clinical trials that may move this field forward to redefine immunologically "hot/cold" tumors and develop next-generation immunotherapeutic approaches.

Conjugation mediates large-scale chromosomal transfer in Streptomyces driving diversification of antibiotic biosynthetic gene clusters.

Streptomyces are ubiquitous soil dwelling bacteria with large, linear genomes that are of special importance as a source of metabolites used in human and veterinary medicine, agronomy and industry. Conjugative elements (Actinomycetes Integrative and Conjugative Elements, AICEs) are the main drivers of Streptomyces Horizontal Gene Transfer. AICE transfer has long been known to be accompanied by mobilization of chromosomal DNA. However, the magnitude of DNA transfer, or the localization of acquired DNA across their linear chromosome, has remained undetermined. We here show that conjugative crossings in sympatric strains of Streptomyces result in the large-scale, genome-wide distributed replacement of up to one third of the recipient chromosome, a phenomenon for which we propose the name 'Streptomyces Chromosomal Transfer' (SCT). Such chromosome blending results in the acquisition, loss and hybridization of Specialized Metabolite Biosynthetic Gene Clusters, leading to a novel metabolic arsenal in exconjugant offspring. Harnessing conjugation-mediated SMBGC diversification holds great promise in the discovery of new bioactive compounds including antibiotics.

Deciphering Plasmodium Condensin Core Subunits of Structural Maintenance of Chromosomes 2 (SMC2) as a Putative Drug Target for Antimalarial Drug

Background: The structural maintenance of chromosomes (SMC) proteins plays a noteworthy role in chromosome dynamics. Several recent studies reported that the condensin core subunits of structural maintenance of chromosomes 2 (SMC2) play important roles in the atypical mitosis of the Plasmodium life cycle and may perform different functions during different proliferative stages. For eukaryotes, the structural maintenance of chromosomes (SMC) proteins are divided into six subunits and form three heterodimers of structural maintenance of chromosomes (SMC1/3) cohesion complex, structural maintenance of chromosomes (SMC2/4) condensin complex, and structural maintenance of chromosomes (SMC5/6) complex for chromosome cohesion, condensation, and DNA damage repair, respectively. Objective: The objective of this study was to investigate the structural maintenance of chromosomes 2 (SMC2) protein of P. falciparum as a putative drug target of malariacausing Plasmodium falciparum. Methods: In this study, we investigated the structural maintenance of chromosomes 2 (SMC2) protein of P. falciparum as a putative drug target of malaria-causing P. falciparum by using in-silico approaches like Homology modeling, in-silico evaluation of the modeled structure, molecular docking study to investigate the interaction of receptor-ligand, and molecular dynamic simulation study with MM calculation. Results: We reported the structural maintenance of chromosomes 2 (SMC2) protein of P. falciparum as a potent drug target that can pave the way for novel drug discovery to mitigate malaria. Conclusion: In-silico-based studies play a significant role in understanding any protein for potential drug development.

Methylome of circulating cell free DNA as a novel biomarker tool: from acute coronary syndrome to broader risk stratification of cardiovascular disease

Abstract Background Cell death during tissue injury leads to release of chromosomal DNA which is promptly degraded. However, short DNA fragments wrapped around nucleosomes survive longer in the bloodstream and can be isolated as circulating cell-free DNA (ccfDNA). Oncology field pioneered the use of cfDNA as clinical biomarker by identifying sequence modifications induced in cancer cells. Importantly, ccfDNA also retains the same epigenetic information as the tissue of origin. Despite major advances for disease diagnosis and therapy, coronary artery diseases (CAD) and associated conditions such acute myocardial infarction (AMI) or heart failure (HF) are responsible for leading number of deaths worldwide. Preventive and surveillance strategies with non-invasive testing are needed. Purpose Evaluation of ccfDNA methylation profiles as prognostic non-invasive biomarker for risk stratification of acute coronary syndromes and their potential application in preventive and surveillance strategies in other cardiovascular disorders. Methods CcfDNA isolated from citrate-plasma collected from our discovery cohort, comprising healthy controls, STEMI, NSTEMI and unstable angina (UA) patients, was analyzed applying Whole Genome Bisulfite Sequencing (WGBS) using a low-input BS-seq (PBAT) protocol. Computational analysis generated differentially methylated regions (DMRs) by tiling CpG areas and setting a threshold of minimal 25% methylation difference compared to the healthy group. To assess the validity of our findings, a selection of disease-specific DMRs were tested using targeted methylation sequencing protocol in a new set of patients. This procedure consisted in enzymatic conversion of ccfDNA methylated sites followed by probe-specific enrichment libraries and sequencing at a Novaseq 6000 platform. Results The study identified a total of 1941 DMRs and from those 486 were STEMI, 223 NSTEMI and 684 UA specific. From the identified DMRs, 74.13% from STEMI, 72.68% from NSTEMI and 61.62% from UA annotated with heart-related diseases using DisGeNET gene-disease associations. Interestingly, most of identified DMRs located within intronic regions (~60%) and also annotated as enhancers and lncRNAs. The independent validation with targeted methylation sequencing identified 566, 306 and 248 DMRs, respectively, and reduced the number of disease-specific DMRs to 171 in STEMI, 115 in NSTEMI and 82 in UA. Conclusion and Future Approaches Methylation profiles of ccfDNA showed potential as a biomarker to stratify the severity of acute coronary syndromes, a novel non-invasive detection methodology that could be extrapolated to risk stratification in other cardiovascular disease such as heart failure or coronary artery disease.

An integrated view of the structure and function of the human 4D nucleome.

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

Structural Characterization of DNA from Allium Leaves and E. coli by Surface-Enhanced Raman Spectroscopy (SERS)

The label-free detection of biological molecules was demonstrated for different types of DNA. Surface-enhanced Raman spectroscopy (SERS) investigation on nucleic acids extracted from leaves of different Allium cepa cultivars (cvs.) De Buzău, Aurie de Buzău, Rubiniu, Roşie de Arieş as well as taxa A. ursinum, A. senescens subsp. montanum, A. schoenoprasum, A. obliquum, and A. fistulosum was performed using 532-nm laser excitation. Main SERS vibrations of these nucleic acids have been characterized, highlighting those at 658, 704, 1089, and 1125 cm−1 attributed to dG and dA phosphodiester groups as well as deoxyribose, specific constituents of DNA. The mild basic pH conditions of the samples can induce a partial breaking of the hydrogen bonds from the dsDNA, which affects the A = T/T = A as well as C≡G/G≡C base pairs and causes the aggregation of AgNPs mixed with DNA of the Allium cultivars and species. Also, SERS profiles of genomic DNA from E. coli 1559 were analyzed at decreasing pHs from 7 to 4. Protonation of the nitrogen N3 of the aromatic six-membered ring of both deoxyadenosine and deoxyguanosine, respectively, induces the decrease of the peak’s intensity at 1588 cm-1. Furthermore, SERS characteristics of plasmidic DNA from E. coli 7832 and of chromosomal DNA from E. coli MG 1655 are provided, with specific SERS modes indicated and new SERS bands found upon nucleic acids in vitro aging. Principal component analysis (PCA) in the full SERS spectrum range of bacterial DNA was performed.

Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger

BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

DNA fragmentation factor 40-based therapeutic approaches for cancer: a review article.

DNA Fragmentation Factor (DFF) is a heterodimer protein involved in DNA fragmentation during apoptosis, which acts as a trigger downstream of caspase-3 activation. DFF40 catalytically active homo-oligomers break down chromosomal DNA. Previous scientific investigations have revealed a link between DFF40 expression changes and various cancers. DFF40 deletion or down-regulation has been observed in some cancers. Consequently, therapeutic strategies involving the DFF40 molecule compensating led to an increased rate of cancer cell apoptosis. In this review article, we aimed to introduce cancers with low expression of this protein first. The second part of this paper focuses on studies that utilized exogenous DFF40 protein produced by recombinant DNA technology and surveyed during in vitro and in vivo tests. Finally, compensation for diminished expression of the mentioned protein via gene therapy-based techniques to make up for this apoptotic molecule's low expression is the topic of thelast part of this review article.