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With the help of the one-dimensional random Potts-like model, we study the origins of fine structures observed on differential melting profiles of double-stranded DNA. We theoretically assess the effects of sequence arrangement on DNA melting curves through the comparison of results for random, correlated, and block sequences. Our results re-confirm the smearing out of the fine structure with the increase in chain length for all types of sequence arrangements and suggest that the fine structure is a finite-size effect. We have found that the fine structures on melting curves of chains comprised of blocks with correlations in sequence are more persistent, probably because of increased sequence disorder the blocks introduce. Many natural DNAs show a well-expressed fine structure of melting profiles. Our results for block sequences may suggest the existence of such sequence motifs in natural DNA sequences. [ABSTRACT FROM AUTHOR]

Species substitution in meat products is a common form of food fraud that raises ethical, religious, dietary, and conservation issues. Measures to monitor meat substitution are being implemented in many parts of the world. However, information on similar efforts in Tanzania is scarce. This study aimed to assess species authenticity in kebabs sold in the Kilosa District. During 4 months (November 2023 to March 2024), 100 processed kebabs were purchased from fast food outlets across 3 wards: Dumila (n = 49), Kimamba (n = 24), and Mikumi (n = 27). Species authentication was conducted using High-resolution melting analysis and DNA barcoding, targeting two mitochondrial markers: cytochrome oxidase 1 (COI) and 16 S rRNA. Out of 100 kebabs, 64 were misidentified. Notably, goat-based kebabs were the most substituted, with 36 out of 43 (83.7%) containing different species: cattle (30), chicken (2), impala (1), and warthog (3). Out of 40 cattle kebabs, 18 (45%) consisted of meat from different species: chicken (7), goat (8), 2 impala (2), and 1 warthog (1). Chicken kebabs had only 7 undeclared cattle species, and all 3 samples sold as mutton kebabs were substituted with cattle (2) and goat (1). The findings indicate a high prevalence of species substitution in meat products within the Kilosa District. Undeclared wildlife species might represent illegal bushmeat harvests in the conserved ecosystems. Extensive meat inspections and educational interventions are recommended to ensure the integrity of the meat market, consumer trust, and the conservation of wildlife. [ABSTRACT FROM AUTHOR]

DNA denaturation, the process of separating double-stranded DNA into single strands, plays a critical role in fundamental biological processes such as transcription, replication, and repair. Despite extensive studies on its thermodynamic properties, the effects of thermal fluctuations on DNA denaturation have not yet been explored. This paper addresses this gap by developing a statistical mechanical model to analyze homogeneous DNA denaturation thermodynamics with thermal fluctuations. Using the partition function framework, this study introduces two major corrections to the entropy of the system induced by thermal fluctuations: (1) a logarithmic correction of the leading order and (2) a higher-order correction term proportional to the inverse of the entropy. Analytical calculations and numerical analysis reveal that these corrections significantly influence the thermodynamic properties, including specific heat and free energy, leading to a more nuanced understanding of the DNA melting process. The corrected entropy modifies the specific heat profile, resulting in a sharp peak that reflects a first-order phase transition during DNA denaturation. The inclusion of higher-order corrections introduces asymmetry in the specific heat curve, highlighting the cooperative behavior of DNA melting. Furthermore, the free-energy analysis suggests the presence of intermediate states during strand separation, which are critical for understanding the initiation and propagation of the denaturation process. The results align well with experimental DNA melting profiles, particularly in the transition region, and provide insights into the microscopic mechanisms underlying DNA melting. This study not only advances the theoretical framework for DNA denaturation by explicitly incorporating thermal fluctuations but also offers a platform for future experimental validation and applications in biological systems. These findings have broader implications for understanding DNA stability under physiological conditions, cellular processes such as transcription initiation, and the role of ionic environments in modulating DNA thermodynamics. [ABSTRACT FROM AUTHOR]

In this study we derive analytically the equilibrium melting probabilities for basepairs of a DNA molecule with a defect site. We assume that the defect is characterized by a change in the Watson–Crick basepair energy of the defect basepair, and in the associated two stacking energies for the defect, as compared to the remaining parts of the DNA. The defect site could, for instance, occur due to DNA basepair mismatching, cross-linking, or by the chemical modifications when attaching fluorescent labels, such as fluorescent-quencher pairs, to DNA. Our exact solution of the Poland–Scheraga model for DNA melting provides the probability that the labeled basepair, and its neighbors, are open at different temperatures. Our work is of direct importance, for instance, for studies where fluorophore-quencher pairs are used for studying single basepair fluctuations of designed DNA molecules. [ABSTRACT FROM AUTHOR]

Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective in genome editing. However, many differences exist in their in vitro dsDNA cleavage activities, reflecting the diversity in Cas12e's enzymatic properties. To comprehensively understand the Cas12e family, we identify and characterize six unreported Cas12e members that vary in their CRISPR-locus architectures, PAM preferences, and cleavage efficacies. Interestingly, among all variants, PlmCas12e exhibits the most robust trans-cleavage activity and the lowest salt sensitivity in cis-cleavage. Further structural comparisons reveal that the unique NTSB domain in PlmCas12e is beneficial to DNA unwinding at high salt concentrations, while some NTSB-lacking Cas12e proteins rely on positively charged loops for dsDNA unwinding. These findings demonstrate how divergent evolution of structural elements shapes the nuclease diversity within the Cas12e family, potentially contributing to their adaptations to varying environmental conditions. CRISPR-Cas12e systems exhibit robust genome editing capabilities, yet their phylogenetic diversity remains underexplored. Here, authors elucidate the biochemical diversity and structural variations of newly identified Cas12e proteins, offering critical insights into structure-activity relationships. [ABSTRACT FROM AUTHOR]

Introduction: Detection of PIK3CA mutations in primary tumors and liquid biopsy samples is of increasing importance for treatment decisions and therapy resistance in many types of cancer. The aim of the present study was to directly compare the efficacy of a relatively inexpensive ultrasensitive real-time PCR with the well-established and highly sensitive technology of ddPCR for the detection of the three most common hotspot mutations of PIK3CA , in exons 9 and 20, that are all of clinical importance in various types of cancer. Patients and methods: We analyzed 42 gDNAs from primary tumors (FFPEs), 29 plasma-cfDNA samples, and 29 paired CTC-derived gDNAs, all from patients with ER+ metastatic breast cancer, and plasma from 10 healthy donors. The same blood draws were used for CTC isolation using EpCAM beads for positive immunomagnetic enrichment. All FFPEs and plasma-cfDNA samples were analyzed in parallel for PIK3CA mutations by ultrasensitive real-time PCR assay and droplet digital PCR. Results: In gDNAs from FFPEs, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 22/42(52.4%), and the p.E542K and p.H1047R mutations were detected in 14/42(33.3%) and 16/42(38.1%), respectively. Using ddPCR, the p.E545K mutation was detected in 22/42(52.4%), p.E542K in 17/42(40.5%), and p.H1047R in 19/42(45.2%) samples, revealing a concordance between the two methodologies of 81%, 78.6% and 78.6% for each mutation respectively. In plasma-cfDNA, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 11/29(38%) and both p.E542K and p.H1047R mutations in 2/29(6.9%).In the same plasma-cfDNA samples using ddPCR, p.E545K was detected in 1/29(3.5%), p.E542K in 2/29(6.9%), and p.H1047R in 3/29(10.5%) samples, revealing a concordance of 65.5%,100% and 93.1% for each mutation respectively. In paired CTC-derived gDNAs p.E545K was detected in 11/29(38%), p.E542K in 3/29(10.3%), and p.H1047R in 7/29(24.1%) samples. Conclusions: This low-cost, high-throughput and ultrasensitive real-time PCR assay provides accurate and specific detection of PIK3CA hotspot mutations in liquid biopsy samples and gives similar results to ddPCR. This assay can be performed in labs where digital PCR instrumentation is not available. In CTC-derived gDNA and paired plasma-cfDNA, PIK3CA mutations detected were not identical, revealing that CTC and plasma-cfDNA give complementary information. [ABSTRACT FROM AUTHOR]

TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungus Ustilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed that UmTrf2 binds to Blm in vitro and inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activity in vivo, over-expression of Trf2 induces telomere shortening, just like deletion of blm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion of blm or mre11 (but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably, U. maydis harbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion of tay1 partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres. Author summary: The ends of linear chromosomes are protected from abnormal repair by a collection of telomere proteins. One protein that plays an especially prominent role is TRF2, which binds to double-stranded telomere repeats. In this study, we analyzed the mechanisms and functions of Trf2 in a yeast-like fungus named Ustilago maydis, which manifests a high degree of similarity to animal cells with respect to telomere regulation. We showed that Trf2 binds directly to a conserved DNA helicase called Blm and inhibits the ability of Blm to unwind telomeric DNA in a purified, cell-free reaction. We also used over-expression and depletion of either Trf2 or Blm or both to demonstrate an inhibitory effect of Trf2 on Blm function in vivo. For example, depletion of Trf2 triggers Blm-dependent telomere aberrations and cell death. Interestingly, another double-strand telomere binding protein named Tay1 was found to stimulate Blm activity to promote telomere replication. Together, our results indicate that U. maydis optimizes Blm function through opposing regulation of its activity via distinct telomere proteins. We also detected high levels of abnormal transcripts that correspond to both strands of telomeres in a variety of telomere mutants, suggesting that de-protected telomeres are permissive substrates for the transcription apparatus. [ABSTRACT FROM AUTHOR]

The spectroscopic investigation of the binding properties of berberine‐type 9‐aryl‐substituted isoquinolinium derivatives with G‐quadruplex DNA (G4‐DNA) are presented. Photometric titrations show that these ligands bind with high affinity to the telomeric G4‐DNA form 22AG (K=1.0–44×105 M−2). Furthermore, fluorimetric analysis of thermal DNA denaturation (FRET melting) reveals a significant thermal stabilization of G4‐DNA 22AG upon association with the methoxy‐substituted derivatives. As an analytically useful property, the derivatives with a phenyl substituent or with additional electron‐donating groups show a very weak fluorescence intensity, which increased significantly upon G4‐DNA complexation (fluorescence light‐up effect). Additional time‐resolved fluorescence spectroscopy indicated increased fluorescence lifetimes of the DNA‐bound 9‐(4‐methoxyphenyl)‐substituted derivative, when interacting with the quadruplex‐forming strand 22AG. Notably, the changes of the steady‐state and time‐resolved emission properties of the ligand are more pronounced with G4‐DNA than with duplex DNA so that the combination of these complementary methods may be used for the selective G4‐DNA detection. [ABSTRACT FROM AUTHOR]

The effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant strands are collected through one specially designed magnetic bead separation in alkaline conditions, efficiently removing their complementary partner strands with the original sequences. In a proof-of-concept test, a green fluorescent protein (GFP) was simultaneously mutated in 1–3 specific amino acids, successfully shifting its fluorescence spectrum. The precise mutation rates for single-, double- and triple-site mutations reached 100%, 76% and 70%, respectively. This multiple non-contiguous mutagenesis method may offer a fast and cost-effective approach for customizable construction of gene library. [ABSTRACT FROM AUTHOR]

The article in the journal "Medcomm: Biomaterials & Applications" discusses how the WED domain in the CRISPR‐Cas9 system plays a crucial role in accelerating DNA unwinding and R‐loop formation, thereby enhancing gene editing efficiency. The study shows that mutations in the WED domain improve Cas9 enzymatic activity and relax PAM recognition preference, leading to more effective genome editing. By understanding the structural and functional relationship of the WED domain, researchers can optimize Cas9 for better gene editing tools, especially in mammalian cells with low magnesium ion concentrations. Future research may focus on enhancing Cas9 stability and expanding target DNA sequences, as well as integrating other gene editing technologies for increased diversity and flexibility. [Extracted from the article]

DNA is considered as a prospective candidate for the next‐generation data storage medium, due to its high coding density, long cold‐storage lifespan, and low energy consumption. Despite these advantages, challenges remain in achieving high‐fidelity, fully integrated, and cost‐efficient DNA storage system. In this study, a homemade digital microfluidic (DMF)‐based compact DNA data storing pipeline is orchestrated to complete the entire process from the synthesis to the sequencing. The synthetic half employs phosphoramidite chemistry on 200 nm magnetic beads (MBs), where the dimethyltrityl protecting group is removed by droplet manipulation of trichloroacetic acid. The sequencing counterpart relies on pyrophosphate releasing originated from polymerase‐catalyzed primer extension, which leads to photon‐countable chemiluminescence (CL) signal in 2.5‐μL drops of trienzyme cascading reactions. Further by DNA denaturation, repeated pyrosequencing plus plurality voting can improve the nucleobase accuracy beyond 95 %. As a proof‐of‐concept trial, semantic information is saved in DNA via the Huffman coding algorithm plus the Reed‐Solomon error‐correction, and then robustly retrieved from this streamlined platform. As a result, it took a net total of approximately 6.5 h to writing and reading 8 bytes of data, that equal to a storaging speed of 49 min/byte, much quicker than the previously reported 2.8–4.2 h/byte. This bead‐based miniaturized device promises an unattended protocol for achieving high‐throughput, full‐packaged, and above all, neatly precision DNA storage. [ABSTRACT FROM AUTHOR]

AbstractThe potential of neurotransmitters and neural hormones as possible G-quadruplex DNA binders was analyzed using fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), DNA melting analysis, and molecular docking. G-quadruplex sequences, (GGC)3 and G4C2, with roles in Fragile X syndrome and amyotrophic lateral sclerosis (ALS), respectively, were selected, and their interactions with melatonin, serotonin, and gamma-aminobutyric acid (GABA), were studied. Both melatonin and serotonin demonstrated strong interactions with the DNA sequences with hydrogen bonding being the primary mode of interaction, with some non-intercalative interactions involving the π systems. GABA demonstrated much weaker interactions and may not be a suitable candidate as a probe for low concentrations of G-quadruplex DNA. [ABSTRACT FROM AUTHOR]

DNA‐binding agents often exhibits dual behavior of simultaneous binding with protein associated with cancerous cell development pathways. This simultaneous protein binding if contributed to malfunctioning of pro‐apoptotic proteins will reduce anticancerous potential of drugs. The elucidation of binding target for anticancerous agent can give an insight into the cancer cell apoptotic pathway. Therefore, anticancerous Zn(II) and Ni(II) complexes of 2‐guanidinobenzimidazole (2GBz) were subjected to DNA‐binding mode assay along with protein‐binding interference assay. The stoichiometry Zn‐2GBz and Ni‐2GBz were determined through density functional theory (DFT) and UV–Vis spectroscopy, followed by structural verification by X‐ray crystallography. The 2GBz and Ni‐2GBz were found to bind with grooves of DNA while groove binding of Zn‐2GBz induced unwinding of DNA through interactive pi‐stacking. Binding constant revealed the M‐2GBz to be a strong binder of DNA molecule with the effect of enhanced cell killing potential of M‐2GBz against MCF‐7 cell lines. Protein‐binding assay revealed that despite of significant interaction of M‐2GBz with chick serum albumin, DNA binding was not altered by simultaneous protein binding. The most potent Zn‐2GBz was found to be sphingosine 1 kinase 2 (SPhK2) which can be a cause of enhanced cancer cell apoptosis with lesser normal cell apoptosis than standard fluorouracil in MCF‐7 cell lines. Co‐administered Zn‐2GBz increased cancer cell sensitivity towards fluorouracil as potent anticancerous agent. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is the etiological agent of African swine fever, a highly contagious hemorrhagic disease affecting both wild boars and domestic pigs with lethality rates up to 100%. Until now, the most effective measure to prevent an outbreak of ASFV was early detection. In this situation, whole genome sequencing (WGS) allows the gathering of detailed information about the identity and epidemiology of the virus. However, due to the large genome size and complex genome ends, WGS is challenging. Current WGS workflows require either elaborate enrichment methods or are based on tiled PCR approaches, which are susceptible to genetic differences between ASFV strains. To overcome this, we developed a novel approach for WGS of ASFV, using the Phi29 DNA polymerase-based multiple displacement amplification in combination with only seven primers. Furthermore, we applied an alkaline-based DNA denaturation step to significantly increase the number of viral reads, which resolves the near-full genome of ASFV. This novel isothermal WGS approach can be used in authorized laboratories for the genomic epidemiological analysis of ASFV outbreaks caused by different genotypes. [ABSTRACT FROM AUTHOR]

Antibiotic resistance genes (ARGs) present in sewage sludge pose significant environmental and public health challenges. Composting has emerged as a promising method to mitigate these risks by reducing ARGs. This review paper evaluated the current progress in the removal of ARGs through composting, incorporating a bibliometric analysis of 228 publications from January 2010 to January 2024. This review highlights the increasing scholarly interest in this field, with a notable rise in publications since 2010. Key mechanisms identified include the denaturation of proteins and DNA at high temperatures, the adsorption of antibiotics and heavy metals by additives like biochar, and shifts in microbial communities, all contributing to the reduction of ARGs during composting. Despite these findings, challenges remain in achieving consistent ARG removal rates, addressing the potential for ARG regrowth, and understanding horizontal gene transfer post-composting. This review suggests further research into optimizing composting conditions and integrating additional treatment methods to enhance ARG removal and minimize associated risks. [ABSTRACT FROM AUTHOR]

Understanding the spatial organization of genomes within chromatin is crucial for deciphering gene regulation. A recently developed CRISPR–dCas9-based genome labeling tool, known as CRISPR–FISH, allows efficient labeling of repetitive sequences. Unlike standard fluorescence in situ hybridization (FISH), CRISPR–FISH eliminates the need for global DNA denaturation, allowing for superior preservation of chromatin structure. Here, we report on further development of the CRISPR–FISH method, which has been enhanced for increased efficiency through the engineering of a recombinant dCas9 protein containing an ALFA-tag. Using an ALFA-tagged dCas9 protein assembled with an Arabidopsis centromere-specific guide RNA, we demonstrate target-specific labeling with a fluorescence-labeled NbALFA nanobody. The dCas9 protein possessing multiple copies of the ALFA-tag, in combination with a minibody and fluorescence-labeled anti-rabbit secondary antibody, resulted in enhanced target-specific signals. The dCas9–ALFA-tag system was also instrumental in live cell imaging of telomeres in Nicotiana benthamiana. This method will further expand the CRISPR imaging toolkit, facilitating a better understanding of genome organization. Furthermore, we report the successful integration of the highly sensitive tyramide signal amplification method with CRISPR–FISH, demonstrating effective labeling of Arabidopsis centromeres. [ABSTRACT FROM AUTHOR]

The Mpox virus (or Monkeypox virus, MPXV) uses its own encoded proteins to form a replication machine that replicates the viral genome in the host cell cytoplasm, making this machinery a key target for antiviral drug design. The D5 (also known as the OPG117 or E5) protein, a bi‐functional helicase–primase enzyme, is crucial in the MPXV replication machinery and genome uncoating process. Recently, cryo‐electron microscopy (cryo‐EM) structures of D5 in multiple states have been determined. These structures have elucidated the full trajectory of the MPXV D5 helicase–primase as it moves along single‐stranded DNA, providing unprecedented advancements in the molecular dynamics and unwinding mechanism. This structural snapshot describes the structural features of the D5 protein and dissects the broader implications of its pivotal role in MPXV replication. [ABSTRACT FROM AUTHOR]

We report that the eukaryotic replicative helicase CMG (Cdc45-MCM-GINS) is required for differential gene expression in cells produced by asymmetric cell divisions in C. elegans. We found that the C. elegans CMG component, PSF-2 GINS2, is necessary for transcriptional upregulation of the pro-apoptotic gene egl-1 BH3-only that occurs in cells programmed to die after they are produced through asymmetric cell divisions. We propose that CMG's histone chaperone activity causes epigenetic changes at the egl-1 locus during replication in mother cells, and that these changes are required for egl-1 upregulation in cells programmed to die. We find that PSF-2 is also required for the divergence of other cell fates during C. elegans development, suggesting that this function is not unique to egl-1 expression. Our work uncovers an unexpected role of CMG in cell fate decisions and an intrinsic mechanism for gene expression plasticity in the context of asymmetric cell division. Cell fate choices, including developmental cell death, are often driven by asymmetric cell division, though what determinants drive these choices remain unclear. Here they show that the evolutionarily conserved CMG complex, best known for its role in unwinding DNA during chromosome replication, is also required for cell fate divergence during asymmetric cell divisions in C. elegans development. [ABSTRACT FROM AUTHOR]

The Poland–Scheraga model, introduced in the 1970s, is a reference model to describe the denaturation transition of DNA. More recently, it has been generalized in order to allow for asymmetry in the strands lengths and in the formation of loops: the mathematical representation is based on a bivariate renewal process, that describes the pairs of bases that bond together. Here, we consider a disordered version of the model, in which the two strands interact via a potential β V (ω ^ i , ω ¯ j) + h when the ith monomer of the first strand and the jth monomer of the second strand meet. Here, h ∈ R is a homogeneous pinning parameter, (ω i ^) i ≥ 1 and ( ω ¯ j) j ≥ 1 are two sequences of i.i.d. random variables attached to each DNA strand, V (· , ·) is an interaction function and β > 0 is the disorder intensity. In our main result, we find some condition on the underlying bivariate renewal so that, if one takes β , h ↓ 0 at some appropriate (explicit) rate as the length of the strands go to infinity, the partition function of the model admits a non-trivial, disordered, scaling limit. This is known as an intermediate disorder regime and is linked to the question of disorder relevance for the denaturation transition. Interestingly, and unlike any other model of our knowledge, the rate at which one has to take β ↓ 0 depends on the interaction function V (· , ·) and on the distribution of (ω i ^) i ≥ 1 , ( ω ¯ j) j ≥ 1 . On the other hand, the intermediate disorder limit of the partition function, when it exists, is universal: it is expressed as a chaos expansion of iterated integrals against a Gaussian process M , which arises as the scaling limit of the field (e β V (ω ^ i , ω ¯ j)) i , j ≥ 0 and exhibits correlations on lines and columns. [ABSTRACT FROM AUTHOR]

The mechanism used by polyomavirus and other viral SF3 helicases to unwind DNA at replication forks remains unknown. Using AlphaFold2, we have determined the structure of a representative SF3 helicase, the SV40 T-antigen (T-ag). This model has been analyzed in terms of the features of T-ag required for helicase activity, particularly the proximity of the T-ag origin binding domain (OBD) to the replication fork and the distribution of basic residues on the surface of the OBD that are known to play roles in DNA unwinding. These and related studies provide additional evidence that the T-ag OBDs have a role in the unwinding of DNA at the replication fork. Nuclear magnetic resonance and modeling experiments also indicate that protonated histidines on the surface of the T-ag OBD play an important role in the unwinding process, and additional modeling studies indicate that protonated histidines are essential in other SF3 and SF6 helicases. Finally, a model for T-ag's helicase activity is presented, which is a variant of the "rope climber." According to this model, the hands are the N-terminal OBD domains that interact with the replication fork, while the C-terminal helicase domains contain the feet that bind to single-stranded DNA. [ABSTRACT FROM AUTHOR]

The paper presents a physicochemical study of the water-soluble adduct of C60 with l-arginine, including a quantum chemical determination of the stability of isomers, measurement of antiradical activity towards the ABTS radical, determination of the lipophilicity coefficient, particle size distribution in aqueous solutions and calculation of ζ-potentials. A separate part of the article is devoted to the study of the effect of the C60 adduct with l-arginine on DNA in in vitro experiments (determination of the binding constant using isothermal titration calorimetry, study of genotoxicity, DNA melting temperature, effect on DNA circular dichroism spectra). Data are also presented on the biodistribution of the C60 adduct with l-arginine in the organs and tissues of animals and its effect on clinical and biochemical blood parameters. The mutagenic effect of the C60 adduct with l-arginine was determined in vivo on a model of mice abnormal sperm heads. It was shown that the adduct is in a neutral state in physiological solution, has an affinity to both aqueous and lipid phases (logPow = 0.23), and forms a complex with DNA (Ka = 4.59 106 M −1), while it does not have cyto- and genotoxicity in vitro and mutagenicity in vivo. [ABSTRACT FROM AUTHOR]

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes. Research combining molecular dynamics simulations and solid-state nanopore measurements reveals distinct disassembly pathways that may explain the distinct properties and roles of H2A.B nucleosomes in spermatogenesis, embryogenesis, and oncogenesis. [ABSTRACT FROM AUTHOR]

All cellular processes that involve the unwinding of DNA also lead to the systematic shuttling of histones. Histone shuttling across the nuclear membrane is facilitated by a class of proteins known as – histone chaperones. Histone chaperones are classified based on their binding to H3/H4 histones or H2A/H2B histones. During the shuttling process, two types of signals - NLS and NES are recognized by the nuclear transport proteins. However, this is the nuclear transport protein and the mechanism of signal recognition by the protein is still unknown. Thus, in this piece of work, the NLS and NES signals are predicted on important H2A/H2B binding histone chaperones. In addition, cellular localization and potential DNA binding regions of histone chaperones are predicted. Mapping of predicted regions on the histone chaperone's structure suggested that the critical binding regions mainly lie on the disordered region of the histone chaperones. NLS and NES are present in the N- and C-terminal of the histone chaperones. Most histone chaperones contain bipartiate NLS signals. This article sheds light on the crucial aspect that in addition of being directly engaged in nucleosome synthesis and disassembly in vivo, histone chaperone also performs various specific roles via histone binding activity. [ABSTRACT FROM AUTHOR]

Photothermal is a significant solar energy conversion process on Earth, and photothermal‐responsive protocells are a potential model for prebiotic chemistry. Here, polydopamine microcapsules are fabricated via emulsion droplets‐templated interfacial polymerization and investigated their potential as synthetic protocell models for photothermal conversion and utilization. DNAzyme‐mediated dopamine peroxidation and polymerization occur at the water/oil interface of the emulsion droplets, facilitated by a biphasic microfluidic technique, leading to the formation of uniform polydopamine microcapsules, which retain structural integrity upon transfer into a continuous water phase. These microcapsules exhibit size‐dependent semi‐permeability and high photothermal capability due to their broad spectral absorbance. The photothermal‐mediated temperature rise within the microcapsules triggers DNA denaturation and rearrangement, and intermittent photothermal irradiation modulates reversible phase separation of DNA condensates, mimicking membrane‐less organelles. This work suggests that interfacial polymerization fabricated polydopamine capsules are a plausible photothermal protocell model that can demonstrate aspects of primitive abiotic cellularity. [ABSTRACT FROM AUTHOR]

Simple Summary: MCM2-7, evolutionarily conserved and essential for DNA replication, functions as the central factor for the eukaryotic replicative DNA helicase. Here, we summarize the roles of MCM2-7 in the initiation and progression of replication forks, with a particular focus on the assembly of the replication complex and its regulation. We also describe the molecular details of the steps required for the transition from the inactive MCM2-7 double hexamer to an active replication fork. In this review, we summarize the processes of the assembly of multi-protein replisomes at the origins of replication. Replication licensing, the loading of inactive minichromosome maintenance double hexamers (dhMCM2-7) during the G1 phase, is followed by origin firing triggered by two serine–threonine kinases, Cdc7 (DDK) and CDK, leading to the assembly and activation of Cdc45/MCM2-7/GINS (CMG) helicases at the entry into the S phase and the formation of replisomes for bidirectional DNA synthesis. Biochemical and structural analyses of the recruitment of initiation or firing factors to the dhMCM2-7 for the formation of an active helicase and those of origin melting and DNA unwinding support the steric exclusion unwinding model of the CMG helicase. [ABSTRACT FROM AUTHOR]

Simple Summary: The MCM2-7 helicase plays a crucial role in unwinding the double-stranded DNA template during DNA replication. Recent studies indicate that the MCM2-7 helicase subunit Mcm2 also facilitates the transfer of the parental histone H3-H4 tetramer (parH3:H4tet) onto the newly replicated lagging strand. This review highlights recent advances in our understanding of the role of the replication machinery, particularly Mcm2, in epigenetic inheritance. Disruption of this function can affect chromatin stability, DNA repair processes, and cell differentiation. Recycling histone proteins from parental chromatin, a process known as parental histone transfer, is an important component in chromosome replication and is essential for epigenetic inheritance. We review recent advances in our understanding of the recycling mechanism of parental histone H3-H4 tetramers (parH3:H4tet), emphasizing the pivotal role of the DNA replisome. In particular, we highlight the function of the MCM2-7 helicase subunit Mcm2 as a histone H3-H4 tetramer chaperone. Disruption of this histone chaperone's functions affects mouse embryonic stem cell differentiation and can lead to embryonic lethality in mice, underscoring the crucial role of the replisome in maintaining epigenomic stability. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5′ → 3′ direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6–4) pyrimidone (6–4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2–5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants. [ABSTRACT FROM AUTHOR]

Metal-mediated base pairs in DNA double helix molecules open up broad opportunities for biosensors based on DNA clusters with silver due to their low toxicity and applicability in drug design. Despite intensive experimental and computational research, molecular mechanisms of stabilization of a double helix by silver-mediated base pairs are mainly unknown. We conducted all-atom molecular dynamics simulations of a dodecameric DNA double helix (sequence 5′-TAGGTCAATACT-3′-3′ATCCACTTATGA-5′) with either cytosine–cytosine or cytosine–Ag+–cytosine mismatch in the center of the duplex. We extended the previously proposed set of interaction parameters for a silver ion in the silver-mediated pair in order to allow for its dissociation. With this new potential, we studied how the addition of a silver ion could stabilize a DNA double helix containing a single cytosine–cytosine mismatch. In particular, we found out that the helix with cytosine–Ag+–cytosine mismatch has a greater melting temperature than the helix with cytosine–cytosine one. This stabilization effect of the silver ion is in qualitative agreement with experimental data. The central region of the duplex with cytosine–Ag+–cytosine mismatch (unlike with cytosine–cytosine mismatch) is stable enough to prevent bubble formation at moderate temperatures during melting. The results of this simulation can be used to devise novel metal-mediated DNA structures. [ABSTRACT FROM AUTHOR]

(1) Background: The detection of methylated SEPT9 (mSEPT9) in plasma is a promising approach to non-invasive colorectal cancer (CRC) screening. Traditional approaches have limitations in sensitivity and cost-effectiveness, particularly in resource-limited settings. (2) Methods: We developed a semi-nested realtime PCR assay utilizing extendable blocking probes (ExBP) to enhance the detection of low-level mSEPT9 based on DNA melting. This assay allows for the discrimination of mSEPT9 in the presence of high concentrations of non-methylated SEPT9 (up to 100,000 times higher). (3) Results: The assay demonstrated a sensitivity of 73.91% and specificity of 80%, showcasing its ability to detect very low levels of methylated DNA effectively. The innovative use of ExBP without costly modified probes simplifies the assay setup and reduces the overall costs, enhancing its applicability in diverse clinical settings. (4) Conclusions: This novel assay significantly improves the detection of mSEPT9, offering a potential advance in CRC screening and monitoring. Its cost-efficiency and high sensitivity make it particularly suitable for the early detection and management of CRC, especially in settings with limited resources. Future studies are encouraged to validate this assay in larger populations to establish its clinical benefits and practical utility. [ABSTRACT FROM AUTHOR]

Melting of DNA sequences may occur through a few major intermediate states, whose influence on the melting curve has been discussed previously, while their effect on the kinetics has not been explored thoroughly. Here, we chose a simple DNA sequence, forming a hairpin in its native (zipped) state, and study it using molecular dynamic (MD) simulations and a model integrating the Gaussian network model with bond-binding energies—the Gaussian binding energy (GBE) model. We find two major partial denaturation states, a bubble state and a partial unzipping state. We demonstrate the influence of these two states on the closing–opening base pair dynamics, as probed by a tagged bond auto-correlation function (ACF). We argue that the latter is measured by fluorescence correlation spectroscopy experiments, in which one base of the pair is linked to a fluorescent dye, while the complementary base is linked to a quencher, similar to the experiment reported by Altan-Bonnet et al. [Phys. Rev. Lett. 90, 138101 (2003)]. We find that tagging certain base pairs at temperatures around the melting temperature results in a multi-step relaxation of the ACF, while tagging other base pairs leads to an effectively single-step relaxation, albeit non-exponential. Only the latter type of relaxation has been observed experimentally, and we suggest which of the other base pairs should be tagged in order to observe multi-step relaxation. We demonstrate that this behavior can be observed with other sequences and argue that the GBE can reliably predict these dynamics for very long sequences, where MD simulations might be limited. [ABSTRACT FROM AUTHOR]

Mpox virus (MPXV) can cause mpox in humans. Due to its quick and wide spread in the past two years, mpox has turned into a significant public health concern. Helicase E5 is a multi-domain protein; its primer synthesis and DNA unwinding activity are required for genome uncoating and DNA replication of MPXV. However, the in vitro DNA unwinding activity has never been demonstrated. Here, we report the structural and biochemical studies of MPXV E5, showing that the full-length protein adopts an auto-inhibited conformation. Truncation of the N-terminus can recover the in vitro unwinding activity of E5 towards the forked DNA. Further structural analysis reveals that MPXV E5 shares a conserved mechanism in DNA unwinding and primer synthesis with the homologous proteins. These findings not only advance our understanding on the function of MPXV E5, but also provide a solid basis for the development of anti-poxvirus drugs. [ABSTRACT FROM AUTHOR]

The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine. [ABSTRACT FROM AUTHOR]

Indole-3-propionic acid (IPA) is a plant growth regulator with good specificity and long action. IPA may be harmful to human health because of its accumulation in vegetables and fruits. Therefore, in this study, the properties of the interaction between calf thymus DNA (ctDNA) and IPA were systematically explored using multispectroscopic and computational modeling approaches. Analysis of fluorescence spectra showed that IPA binding to ctDNA to spontaneously form a complex was mainly driven by hydrogen bonds and hydrophobic interaction. DNA melting analysis, viscosity analysis, DNA cleavage study, and circular dichroism measurement revealed the groove binding of IPA to ctDNA and showed that the binding did not significantly change ctDNA confirmation. Furthermore, molecular docking found that IPA attached in the A-T rich minor groove region of the DNA. Molecular dynamics simulation showed that DNA and IPA formed a stable complex and IPA caused slight fluctuations for the residues at the binding site. Gel electrophoresis experiments showed that IPA did not significantly disrupt the DNA structure. These findings may provide useful information on the potential toxicological effects and environmental risk assessments of IPA residue in food at the molecular level. [ABSTRACT FROM AUTHOR]

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques. [ABSTRACT FROM AUTHOR]

The document is a contents list for the journal Chemical Communications, published by The Royal Society of Chemistry. It includes a feature article on the use of tetrazoles in constructing assemblies, as well as several communications on various topics such as electron-poor Au(III) trication, cold denaturation of DNA origami nanostructures, and optical discrimination of terpenes in citrus peels. The journal aims to connect the global chemistry community and publishes work from scientists at all career stages and countries. [Extracted from the article]

The coupling of structural transitions to heat capacity changes leads to destabilization of macromolecules at both elevated and lowered temperatures. DNA origami not only exhibit this property but also provide a nanoscopic observable of cold denaturation processes by directing intramolecular strain to the most sensitive elements within their hierarchical architecture. [ABSTRACT FROM AUTHOR]

Surveillance for genetic variation of microbial pathogens, both within and among species, plays an important role in informing research, diagnostic, prevention, and treatment activities for disease control. However, large-scale systematic screening for novel genotypes remains challenging in part due to technological limitations. Towards addressing this challenge, we present an advancement in universal microbial high resolution melting (HRM) analysis that is capable of accomplishing both known genotype identification and novel genotype detection. Specifically, this novel surveillance functionality is achieved through time-series modeling of sequence-defined HRM curves, which is uniquely enabled by the large-scale melt curve datasets generated using our high-throughput digital HRM platform. Taking the detection of bacterial genotypes as a model application, we demonstrate that our algorithms accomplish an overall classification accuracy over 99.7% and perform novelty detection with a sensitivity of 0.96, specificity of 0.96 and Youden index of 0.92. Since HRM-based DNA profiling is an inexpensive and rapid technique, our results add support for the feasibility of its use in surveillance applications. [ABSTRACT FROM AUTHOR]

This document is an editorial from the International Journal of Molecular Sciences titled "Chemistry and Biology of Noncanonical Nucleic Acid Structures: From Physicochemical Properties to Therapeutic Applications." The editorial aims to highlight the chemistry and biology of noncanonical nucleic acid structures, focusing on their structure, stability, and interactions with proteins and ligands. Noncanonical structures, such as triplex, G-quadruplex, and i-motif species, can form in important genomic regions and play key roles in biological processes, including oncogene expression and telomere maintenance. The investigations published in this special issue cover various aspects, including the potential role of noncanonical structures in transcription regulation, the selective targeting of cancer-related G-quadruplex structures, and the development of innovative analytical methods for the detection of viral RNA. The document concludes by emphasizing the potential of noncanonical nucleic acids for therapeutic applications and the need for further research in this field. [Extracted from the article]

Torsional stress in double-stranded DNA enables and regulates facets of chromosomal metabolism, replication, and transcription and requires regulatory enzymatic systems including topoisomerases and histone methyltransferases. As such, this machinery may be subject to deleterious effects from reactive mutagens, including ones from carcinogenic polycyclic aromatic hydrocarbon (PAH) adduct formation with DNA. Supercoiled plasmid DNA was investigated for its torsional responses to adducts formed in vitro from PAH benzylic carbocation reactive intermediates created spontaneously by release of leaving groups. PAH sulfate esters were found to (1) unwind DNA in a concentration dependent manner, and (2) provide maximum unwinding in a pattern consistent with known carcinogenicities of the parent PAHs, that is, 6-methylbenzo[a]pyrene > 7,12-methylbenz[a]anthracene > 3-methylcholanthrene > 9-methylanthracene > 7-methylbenz[a]anthracene > 1-methylpyrene. Supercoil unwinding was demonstrated to be dependent on the presence of sulfate or chloride leaving groups such that reactive carbocations were generated in situ by hydrolysis. In silico modeling of intercalative complex topology showed PAH benzylic carbocation reactive functional groups in alignment with target nucleophiles on guanine bases in a 5'-dCdG-3' pocket in agreement with known formation of nucleotide adducts. Inhibitory or modulatory effects on PAH-induced supercoil unwinding were seen with ascorbic acid and an experimental antineoplastic agent Antineoplaston A10 in agreement with their known anticarcinogenic properties. In summary, the reactive PAH intermediates studied here undoubtedly participate in well-known mutational mechanisms such as frameshifts and apurinic site generation. However, they are also capable of random disruption of chromosomal supercoiling in a manner consistent with the known carcinogenicities of the parent compounds, and this mechanism may represent an additional detrimental motif worthy of further study for a more complete understanding of chemical carcinogenicity. [ABSTRACT FROM AUTHOR]

The thermal stability of DNA immobilized on a solid surface is one of the factors that affects the efficiency of solid-phase amplification (SP-PCR). Although variable temperature amplification ensures high specificity of the reaction by precisely controlling temperature changes, excessively high temperatures during denaturation can negatively affect DNA stability. Formamide (FA) enables DNA denaturation at lower temperatures, showing potential for SP-PCR. Research on FA's impacts on DNA microarrays is still limited, necessitating further optimization in exploring the characteristics of FA in SP-PCR according to particular application needs. We immobilized DNA on a chip using a crosslinker and generated DNA microarrays through bridge amplification based on FA denaturation on our automated reaction device. We optimized the denaturation and hybridization parameters of FA, achieving a maximum cluster density of 2.83 × 104 colonies/mm2. Compared to high-temperature denaturation, FA denaturation required a lower template concentration and milder reaction conditions and produced higher cluster density, demonstrating that FA effectively improves hybridization rates on surfaces. Regarding the immobilized DNA stability, the FA group exhibited a 45% loss of DNA, resulting in a 15% higher DNA retention rate compared to the high-temperature group, indicating that FA can better maintain DNA stability. Our study suggests that using FA improves the immobilized DNA stability and amplification efficiency in SP-PCR. [ABSTRACT FROM AUTHOR]

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well- studied pentameric ATPase complex RFC (RFC1- 5). The CTF18- RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo- EM analysis of in vitro assembled human CTF18- RFC--DNA--PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3- ss/dsDNA junction by CTF18- RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique β- hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3- end of the primer; this ensures its ability to load PCNA to any 3- ss/dsDNA junction, facilitated by the binding energy of the E- plug to the major groove. Our study reveals unique structural features of the human CTF18- RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders. [ABSTRACT FROM AUTHOR]

DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide excision repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers from ultraviolet light or DNA crosslinking damage from platinum‐based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10‐subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre‐initiation complex and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG‐NER) and transcription‐coupled NER (TC‐NER). We will also discuss how TFIIH's roles in the two subpathways lead to different genetic disorders. [ABSTRACT FROM AUTHOR]

Simple Summary: Here, we summarize what is known about the non-replication roles of MCM helicase, best known for its ability to unwind double-stranded DNA templates during DNA replication. The MCM2-7 complex is a hexameric protein complex that serves as a DNA helicase. It unwinds the DNA double helix during DNA replication, thereby providing the single-stranded replication template. In recent years, it has become clear that the MCM2-7 complex has additional functions that extend well beyond its role in DNA replication. Through physical and functional interactions with different pathways, it impacts other nuclear events and activities, including folding of the genome, histone inheritance, chromosome segregation, DNA damage sensing and repair, and gene transcription. Collectively, the diverse roles of the MCM2-7 complex suggest it plays a critical role in maintaining genome integrity by integrating the regulation of DNA replication with other pathways in the nucleus. [ABSTRACT FROM AUTHOR]

Simple Summary: During cell division, DNA is duplicated through a process called DNA replication, so that each new cell inherits a copy of its own. A high level of accuracy is essential in this for the maintenance of genome stability and the prevention of genetic disorders and ageing-related diseases. In this review, we describe the current knowledge around DNA replication termination, in particular comparing and contrasting the proteins and mechanisms identified in different organisms—from archaea through to humans—but with a specific focus upon eukaryotic replication termination. We discuss when and where termination takes place, the mechanisms of replication fork convergence and the process of replisome disassembly, in both S-phase and in mitosis. Recent advances in the field have revealed high levels of regulation in the process of replisome disassembly, demonstrating the importance of timely and appropriate unloading of replication machinery. Finally, we summarise how replication termination defects may impact cellular health and raise questions to be addressed in the future within the field. The eukaryotic replicative helicase (CMG complex) is assembled during DNA replication initiation in a highly regulated manner, which is described in depth by other manuscripts in this Issue. During DNA replication, the replicative helicase moves through the chromatin, unwinding DNA and facilitating nascent DNA synthesis by polymerases. Once the duplication of a replicon is complete, the CMG helicase and the remaining components of the replisome need to be removed from the chromatin. Research carried out over the last ten years has produced a breakthrough in our understanding, revealing that replication termination, and more specifically replisome disassembly, is indeed a highly regulated process. This review brings together our current understanding of these processes and highlights elements of the mechanism that are conserved or have undergone divergence throughout evolution. Finally, we discuss events beyond the classic termination of DNA replication in S-phase and go over the known mechanisms of replicative helicase removal from chromatin in these particular situations. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious and pathogenic infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). It manifests as reproductive disorders in sows and respiratory disorders in piglets. PRRSV infects swine herds with symptoms such as abortions, stillbirths, and mummified fetuses in gestating sows. Piglets mainly experience abdominal respiration and respiratory symptoms. To date, the prevention of PRRS relies primarily on vaccination and the implementation of various preventive and control measures. Swine deaths caused by PRRS have resulted in significant economic losses to the pig industry worldwide. Non-structural protein 10 (NSP10) has helicase and adenosine triphosphatase (ATPase) activities that unwind DNA and RNA and play important roles in viral replication and transcription. Hence, it can be potentially used to develop novel reagents for the detection of PPRSV. This article reviews genetic variations, interaction with viral and host proteins, effects on PRRSV replication, immunomodulation, apoptosis, and viral virulence of NSP10, with the aim of providing a theoretical basis for the prevention and control of PRRS and drug development in the future. [ABSTRACT FROM AUTHOR]

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity. The most recent class of base editors utilize DddAtox, a deaminase domain that can act upon double-stranded DNA. Here the authors target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers; they also identify a variety of DddAtox orthologues. [ABSTRACT FROM AUTHOR]