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The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45‐MCM‐GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading‐strand polymerase Pol ε, together with TIMELESS‐TIPIN, CLASPIN and AND‐1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Å cryo‐EM structure of a human replisome comprising CMG, Pol ε, TIMELESS‐TIPIN, CLASPIN and AND‐1 bound to replication fork DNA. The structure permits a detailed understanding of how AND‐1, TIMELESS‐TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS‐TIPIN with replication fork DNA suggests a mechanism for strand separation. SYNOPSIS: Cryo‐EM structures of the core human replisome reveal its complex architecture and show how TIMELESS‐TIPIN, AND‐1, Pol ε and CLASPIN engage the CMG helicase. Contacts between MCM subunits and DNA suggest a mechanism for strand separation. High‐resolution structure of a reconstituted human replisome comprising the CMG helicase, AND‐1, TIMELESS‐TIPIN, Pol ε, CLASPIN and fork DNA.Detailed description of the protein‐protein and protein‐DNA contacts that underpin the organisation of the human replisome.Atomic model for three regions of CLASPIN showing how the protein extends across one side of the replisome contacting TIMELESS, MCM2 and MCM6.Identification of a specific conformation for the catalytic domain of Pol ε, demonstrating that Pol ε can adopt a "linear" configuration in the human replisome. [ABSTRACT FROM AUTHOR]

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases. UvrD is a model helicase from the non-hexameric Superfamily 1. Here, the authors use optical tweezers to measure directly the stepwise translocation of UvrD along a DNA hairpin, and propose a mechanism in which UvrD moves one base pair at a time, but sequesters the nascent single strands, releasing them after a variable number of ATP hydrolysis cycles. [ABSTRACT FROM AUTHOR]

Geminivirus replication initiator protein (Rep) is a multifunctional viral protein required for replication. During the process of viral replication, Rep acts as a site‐ and strand‐specific endonuclease, ligase, ATPase, and helicase. B' motif and β‐hairpin loop of the geminivirus Rep are conserved and important for Rep‐mediated helicase activity required for viral replication. To dissect the roles of various amino acid residues of the B' motif and β‐hairpin loop of the geminivirus Rep helicase in its process of unwinding DNA, we investigated eight conserved residues near the ATP active site or the ssDNA contact channel. Our strategy was to mutate these residues to alanines and investigate the effects of these mutations on various biochemical activities associated with DNA unwinding. We looked into the ATP binding, ATP hydrolysis, DNA binding, and DNA unwinding activities of the wild‐type and mutant Rep proteins. These investigations showed four residues (Arg279, Asp280, Tyr287, and Pro290) affecting the DNA unwinding activity. A structural model analysis confirmed the B' loop and ssDNA binding loop to be connected through a β‐hairpin structure, suggesting that changes on one loop might affect the other and that these residues function by acting in concert. Viral genomes containing Rep proteins having these mutations in the B' motif did not replicate inplanta. Taken together, these results indicated all four residues to be implicated in helicase activity mediated by Rep and demonstrated the significance, for viral replication, of the B' motif and β‐hairpin loop of the C‐terminal region of the Rep protein. [ABSTRACT FROM AUTHOR]

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork. Author summary: DNA replication is a vital biological process, and the proteins involved are structurally and functionally conserved across all domains of life. As our fundamental knowledge of genes and genetics grows, so does our awareness of links between acquired genetic mutations and disease. Understanding how genetic material is replicated accurately and efficiently and with high fidelity is the foundation to identifying and solving genome-based diseases. E. coli are model organisms, containing core replisome proteins, but lack the complexity of the human replication system, making them ideal for investigating conserved replisome behaviors. The helicase enzyme acts at the forefront of the replication fork to unwind the DNA helix and has also been shown to help coordinate other replisome functions. In this study, we examined specific mutations in the helicase that have been shown to regulate its conformation and speed of unwinding. We investigate how these mutations impact the growth, fitness, and cellular morphology of bacteria with the goal of understanding how helicase regulation mechanisms affect an organism's ability to survive and maintain a stable genome. [ABSTRACT FROM AUTHOR]

The main peculiarities of the interaction of DNA and protein molecules with graphite are considered. The results of atomic force microscopy study of the adsorption of DNA and proteins on the surface of highly oriented pyrolytic graphite are analyzed. These data are compared with the results of molecular dynamics simulation and optical studies. The dehybridization of DNA and the denaturation of many proteins on the graphite surface which are predominantly associated with π–π-stacking interaction (for DNA and proteins) and van der Waals and hydrophobic interactions (for proteins) are discussed. It is shown that the surface of highly oriented pyrolytic graphite is strongly liable to random environmental contaminations that considerably change the properties of the surface and, therefore, influence the kinetics of adsorption of biopolymer molecules and their conformation on the surface. Considerable attention is given to analysis of the interaction of biopolymers with the surface of highly oriented pyrolytic graphite modified with monolayers of organic molecules. [ABSTRACT FROM AUTHOR]

We investigate the sharp asymptotic behavior at criticality of the large fluctuations of extensive observables in renewal models of statistical mechanics, such as the Poland–Scheraga model of DNA denaturation, the Fisher–Felderhof model of fluids, the Wako–Saitô–Muñoz–Eaton model of protein folding, and the Tokar–Dreyssé model of strained epitaxy. These models amount to Gibbs changes of measure of a classical renewal process and can be identified with a constrained pinning model of polymers. The extensive observables that enter the thermodynamic description turn out to be cumulative rewards corresponding to deterministic rewards that are uniquely determined by the waiting time and grow no faster than it. The probability decay with the system size of their fluctuations switches from exponential to subexponential at criticality, which is a regime corresponding to a discontinuous pinning–depinning phase transition. We describe such decay by proposing a precise large deviation principle under the assumption that the subexponential correction term to the waiting time distribution is regularly varying. This principle is, in particular, used to characterize the fluctuations of the number of renewals, which measures the DNA-bound monomers in the Poland–Scheraga model, the particles in the Fisher–Felderhof model and the Tokar–Dreyssé model, and the native peptide bonds in the Wako–Saitô–Muñoz–Eaton model. [ABSTRACT FROM AUTHOR]

Background: The role of long noncoding RNA (LncRNA) ADAMTS9 antisense RNA 2 (ADAMTS9-AS2) is unclear in lung adenocarcinoma (LUAD). The aim of this study was to explore the relationship between ADAMTS9-AS2 and LUAD, based on The Cancer Genome Atlas (TCGA) database and bioinformatics analysis. Methods: Various statistical methods, Kaplan–Meier method, Cox regression analysis, GSEA, and immune infiltration analysis were used to evaluate the relationship between clinical features and ADAMTS9-AS2 expression, prognostic factors, and the significant involvement of ADAMTS9-AS2 in function. Results: In LUAD patients, low expression of ADAMTS9-AS2 was associated with N stage (P=0.011), gender (P=0.002), number of packs smoked (P=0.024) and smoker (P< 0.001). Low ADAMTS9-AS2 expression predicted a poorer overall survival (OS) (HR: 0.68; 95% CI: 0.51– 0.91; P=0.01). And ADAMTS9-AS2 expression (HR: 0.626; 95% CI: 0.397– 0.986; P=0.043) was independently correlated with OS in LUAD patients. Unwinding of DNA, extrinsic pathway, polo-like kinase-mediated events, cori cycle, MCM pathway, proteasome pathway, lagging strand synthesis and PCNA-dependent long patch base excision repair were differentially enriched in ADAMTS9-AS2 high expression phenotype. ADAMTS9-AS2 expression was correlated with certain immune infiltrating cells. Conclusion: In LUAD patients, ADAMTS9-AS2 expression was significantly associated with poor survival and immune infiltration. ADAMTS9-AS2 may be a promising biomarker of prognosis and response to immunotherapy for LUAD. [ABSTRACT FROM AUTHOR]

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPo). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyse RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load- and- lock', model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters. [ABSTRACT FROM AUTHOR]

The present study sought to evaluate the interaction between aflatoxin G1 and free DNA in vitro through different analytical techniques. The UV–visible spectra results showed that the structure of DNA might be changed with a new aflatoxin G1-DNA complex forming, which indicated that the interacting mode between them was the intercalating mode. The DNA melting temperature increased by 12.80 °C, suggesting that the DNA double helix structure was more compact and stable through intercalation. The circular dichroism (CD) spectra results indicated that the interaction of aflatoxin G1 with DNA induced the DNA base stacking changes. The results of agarose gel electrophoresis and fluorescence microscope further verified that the interacting mode between aflatoxin G1 and DNA was intercalation mode. According to the fluorescence spectrum data, the binding constant was calculated 6.24 × 104 L·mol−1. The thermodynamic results demonstrated that the reaction of aflatoxin G1 intercalating to DNA was a spontaneous reaction. The elimination results suggested that aflatoxin G1 could be enriched and removed by DNA intercalation through magnetic beads separation, with the removal efficiency of 93.73%. The study results would provide a theoretical basis for establishing a new aflatoxin removal method based on DNA intercalation. [ABSTRACT FROM AUTHOR]

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A trans-versions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended. [ABSTRACT FROM AUTHOR]

Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy. Geminin-Cdt1 plays essential roles in the regulation of DNA replication. Here the authors reveal that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP prevents DNA replication over-firing and genome instability by affecting Geminin ubiquitination. [ABSTRACT FROM AUTHOR]

The present study sought to evaluate the interaction between aflatoxin G1 and free DNA in vitro through different analytical techniques. The UV–visible spectra results showed that the structure of DNA might be changed with a new aflatoxin G1-DNA complex forming, which indicated that the interacting mode between them was the intercalating mode. The DNA melting temperature increased by 12.80 °C, suggesting that the DNA double helix structure was more compact and stable through intercalation. The circular dichroism (CD) spectra results indicated that the interaction of aflatoxin G1 with DNA induced the DNA base stacking changes. The results of agarose gel electrophoresis and fluorescence microscope further verified that the interacting mode between aflatoxin G1 and DNA was intercalation mode. According to the fluorescence spectrum data, the binding constant was calculated 6.24 × 104 L·mol−1. The thermodynamic results demonstrated that the reaction of aflatoxin G1 intercalating to DNA was a spontaneous reaction. The elimination results suggested that aflatoxin G1 could be enriched and removed by DNA intercalation through magnetic beads separation, with the removal efficiency of 93.73%. The study results would provide a theoretical basis for establishing a new aflatoxin removal method based on DNA intercalation. [ABSTRACT FROM AUTHOR]

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts. [Display omitted] • Cryo-EM structures of Fanzor from three organisms in different conformational states • Structures reveal common features and structural diversity of Fanzor • Local conformational changes regulate target DNA manipulation by Fanzor Xu et al. resolve multiple structures of Fanzors (Fzs), eukaryotic ωRNA-guided endonucleases, revealing distinctive features of these proteins found in different organisms and showing how conformational changes regulate the nuclease activity of Fzs. [ABSTRACT FROM AUTHOR]

[Display omitted] • DNA maintains the B-conformation after interacting with ILs ([bmim]Cl and [omim]Cl). • ILs induce unwinding and softening of DNA. • ILs binding to DNA surface reduce DNA torsional stiffness and twist-stretch coupling. • ILs reduce Na+ and water binding on DNA and take part in the solvation process of DNA. The functions of DNA are related to its mechanical properties, including elasticity and conformational transitions. Ionic liquids, known as green solvents, are used for DNA separation and as a solvent medium for long-term DNA storage. This paper elucidated DNA interaction mechanisms with two imidazolium-based ionic liquids: 1-butyl-3-methylimi-dazolium chloride ([bmim]Cl) and 1-octyl-3-methylimidazolium chloride ([omim]Cl), via magnetic tweezers, bulk techniques and molecular dynamic simulation (MD). By stretching and twisting individual DNA molecules, we measured the elasticity of DNA in the presence of ILs ([bmim]Cl and [omim]Cl). As results, the persistence length, stretch modulus, torsional stiffness and twist-stretch coupling exhibited a significant decrease compared to the DNA without ILs binding. Isothermal titration calorimetry analysis indicated that the ILs in the presence of DNA were thermodynamically favored driven by enthalpy and entropy. DNA underwent size transition and conformational change induced by ILs, and the charges of DNA were neutralized by the added ILs. The binding of ILs induced unwinding and softening of DNA. Circular dichroism spectra and MD results indicated that DNA maintained B-conformation after interacting with ILs. Compared to [bmim]+, [omim]+ showed stronger binding affinity to DNA and tended to aggregate on the DNA surface. ILs binding reduced Na+ and water binding on DNA, which likely prevented the hydrolytic reactions that denatured DNA and helped stabilize DNA for the long term. This research provides a critical theoretical foundation for the utilization of ionic liquids in life sciences. [ABSTRACT FROM AUTHOR]

An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5′ to 3′ direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination. • Canonical polymerase roles were defined by several innovative techniques. • The structure of the replisome, built around the helicase, dictates polymerase use. • Pol δ is used flexibly for different replication stages and DNA damage tolerance. • Mapping polymerase usage reveals global replication dynamics. [ABSTRACT FROM AUTHOR]

The contributions of anti-Topoisomerase 1 (Top1) autoantibodies to the pathophysiology of diffuse cutaneous systemic sclerosis (dcSSc), the most aggressive scleroderma subtype, are unknown. Top1 catalyzes DNA relaxation and unwinding in cell nuclei, a site previously considered inaccessible to antibodies. The discovery of autoantibodies in systemic lupus erythematosus that penetrate nuclei and inhibit DNA repair raised the possibility that nuclear-penetrating autoantibodies contribute to mechanisms of autoimmunity. Here we show that an anti-Top1 autoantibody produced by a single B cell clone from a patient with dcSSc penetrates live cells and localizes into nuclei. Functionally, the autoantibody inhibits formation of the Top1 cleavage complex necessary for DNA nicking, which distinguishes it from cytotoxic camptothecin Top1 inhibitors used in cancer therapy that trap the cleavage complex rather than preventing its formation. Discovery of a patient-derived cell-penetrating scleroderma anti-Top1 autoantibody that inhibits Top1 cleavage complex formation supports the hypothesis that anti-Top1 autoantibodies contribute to cellular dysfunction in dcSSc and offers a valuable antibody reagent resource for future studies on anti-Top1 autoantibody contributions to scleroderma pathophysiology. [Display omitted] • Discovery of a nuclear-penetrating human scleroderma anti-Top1 autoantibody. • Nuclear-penetrating anti-Top1 autoantibody blocks Top1 cleavage complex formation. • Findings support a role of cell-penetrating autoantibodies in scleroderma. • A nuclear-penetrating scleroderma anti-Top1 autoantibody reagent is established. [ABSTRACT FROM AUTHOR]

Food adulteration has been an issue in the food industry for many decades. The market for natural and healthy products has been expanding and therefore the value of raw ingredients harvested from natural sources has increased, and, in some cases, it has led to adulteration of such high-valued products. Berry fruits and products from different species of the genus Vaccinium have been gaining worldwide popularity because of their nutraceutical benefits. However, there are increasing reports of adulteration or misidentification of high-value wild berries with their cultivated counterparts. During recent years, DNA-based methods for the authentication of commercial products have gained popularity, especially regarding species identification. Based on the availability of their chloroplast genome information, we developed DNA markers to differentiate lingonberry from cranberry as well as to discriminate European bilberry from North American blueberry. By using Bar-HRM analysis (DNA barcoding combined with High Resolution Melting), the markers were tested on plant DNA as well as DNA extracted from diverse berry products. The developed markers were able to discriminate between species and, moreover, detect potential adulteration in products made from these berry species. Subsequently, the Bar-HRM result was verified by amplicon sequencing. To conclude, the developed molecular markers represent a valuable tool for the berry food industry. • Increasing value of natural ingredients may lead to adulteration of berry products. • Kit-based DNA extraction from berry products was optimized for easier authentication. • DNA markers were developed to discriminate between Vaccinium species by Bar-HRM. • Markers were found to separate blueberry from bilberry and lingonberry from cranberry. • Markers detected potential adulteration of berry products in Bar-HRM analysis. [ABSTRACT FROM AUTHOR]

[Display omitted] • The 5′-WGWWCW-3′ motif serves as the binding site for several nuclear receptors. • Rapid 1D NMR screening tool was utilized to assess ligand binding to the minor groove of DNA. • This study presents the first small-molecular-weight MGBs that bind with 5′-WGWWCW-3′ motif. • A good basis to guide future development of novel MGBs as potential therapeutic agents. Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against the self-complementary DNA sequences 5′-d(CGC ACTAGT GCG)-3′ and 5′-d(CGC AGTACT GCG)-3′. The ligands were carefully designed to target the DNA response element, 5′-WGWWCW-3′, the binding site for several nuclear receptors. Basic 1D 1H NMR spectra of the DNA samples prepared with three MGB ligands show subtle variations suggestive of how each ligand associates with the double helical structure of both DNA sequences. The variations among the investigated ligands were reflected in the line shape and intensity of 1D 1H and 31P-{1H} NMR spectra. Rapid visual inspection of these 1D NMR spectra proves to be beneficial in providing valuable insights on MGB binding molecules. The NMR results were consistent with the findings from both UV DNA denaturation and molecular modelling studies. Both the NMR spectroscopic and computational analyses indicate that the investigated ligands bind to the minor grooves as antiparallel side-by-side dimers in a head-to-tail fashion. Moreover, comparisons with results from biochemical studies offered valuable insights into the mechanism of action, and antitumor activity of MGBs in relation to their structures, essential pre-requisites for future optimization of MGBs as therapeutic agents. [ABSTRACT FROM AUTHOR]

Early-onset ataxia, hearing loss, hypomyelinating, cerebellar atrophy and thin corpus callosum are consistent in our case, which confirms that I RNF220 i is a novel gene related to this combination of phenotypes. A recent study in I Brain i [1] identified two homozygous missense variants in I RNF220 i [p.(R363Q) and p.(R365Q)] in five European pedigrees with ataxia, deafness, hypomyelinating leukodystrophy, liver dysfunction and dilated cardiomyopathy. [Extracted from the article]

In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a laborious manual process, Bionano Genomics platforms now offer high-throughput, automated optical mapping based on chips packed with nanochannels through which unwound DNA is guided and the fluorescent DNA backbone and specific restriction sites are recorded. Although the raw image data obtained is of high quality, the processing and assembly software accompanying the platforms is closed source and does not seem to make full use of data, labeling approximately half of the measured signals as unusable. Here we introduce two new software tools, independent of Bionano Genomics software, to extract and process molecules from raw images (OptiScan) and to perform molecule-to-molecule and molecule-to-reference alignments using a novel signal-based approach (OptiMap). We demonstrate that the molecules detected by OptiScan can yield better assemblies, and that the approach taken by OptiMap results in higher use of molecules from the raw data. These tools lay the foundation for a suite of open-source methods to process and analyze high-throughput optical mapping data. The Python implementations of the OptiTools are publicly available through http://www.bif.wur.nl/. [ABSTRACT FROM AUTHOR]

G-quadruplex DNA interacts with the N-terminal intrinsically disordered domain of the DEAD-box helicase Ded1p, diminishing RNA unwinding activity but enhancing liquid–liquid phase separation of Ded1p in vitro and in cells. The data highlight multifaceted effects of quadruplex DNA on an enzyme with intrinsically disordered domains. [ABSTRACT FROM AUTHOR]

The chromatin structure is dynamically regulated by many different modulators that post-translationally modify histones, replace canonical histones with histone variants, and unwind nucleosomal DNA, thereby modulating the accessibility of nucleosomal DNA and facilitating downstream DNA-templated nuclear processes. To understand how these modulators change the chromatin structure, it is essential to determine the 3D structures of chromatin modulators in complex with nucleosome. Here, we review the very recent progress in structural studies of some selected chromatin modulators in complex with nucleosome, including those of histone demethylases LSD1/2, some pioneer transcription factors, and the PWWP domain–containing protein LEDGF. [ABSTRACT FROM AUTHOR]

The Mre11-Rad50-Nbs1/Xrs2 protein complex plays a pivotal role in the detection and repair of DNA double strand breaks. Through traditional and emerging structural biology techniques, various functional structural states of this complex have been visualized; however, relatively little is known about the transitions between these states. Indeed, it is these structural transitions that are important for Mre11-Rad50-mediated DNA unwinding at a break and the activation of downstream repair signaling events. Here, we present a brief overview of the current understanding of the structure of the core Mre11-Rad50 complex. We then highlight our recent studies emphasizing the contributions of solution state NMR spectroscopy and other biophysical techniques in providing insight into the structures and dynamics associated with Mre11-Rad50 functions. [ABSTRACT FROM AUTHOR]

Synthesis and purification of metal clusters without strong binding agents by wet chemical methods are very attractive for their potential applications in many research areas. However, especially challenging is the separation of uncharged clusters with only a few number of atoms, which renders the usual techniques very difficult to apply. Herein, we report the first efficient separation of Ag2 and Ag3 clusters using the different entropic driving forces when such clusters interact with DNA, into which Ag3 selectively intercalates. After sequential dialysis of the samples and denaturalizing the DNA‐Ag3 complex, pure Ag2 can be found in the dialysate after extensive dialysis. Free Ag3 is recovered after DNA denaturation. [ABSTRACT FROM AUTHOR]

Warsaw breakage syndrome (WABS), is caused by biallelic mutations of DDX11, a gene coding a DNA helicase. We have recently reported two affected sisters, compound heterozygous for a missense (p.Leu836Pro) and a frameshift (p.Lys303Glufs*22) variant. By investigating the pathogenic mechanism, we demonstrate the inability of the DDX11 p.Leu836Pro mutant to unwind forked DNA substrates, while retaining DNA binding activity. We observed the accumulation of patient‐derived cells at the G2/M phase and increased chromosomal fragmentation after mitomycin C treatment. The phenotype partially overlaps with features of the Fanconi anemia cells, which shows not only genomic instability but also defective mitochondria. This prompted us to examine mitochondrial functionality in WABS cells and revealed an altered aerobic metabolism. This opens the door to the further elucidation of the molecular and cellular basis of an impaired mitochondrial phenotype and sheds light on this fundamental process in cell physiology and the pathogenesis of these diseases. [ABSTRACT FROM AUTHOR]

DNA replication complexes (replisomes) frequently encounter barriers that can eject them prematurely from the genome. To avoid the lethality of incomplete DNA replication that arises from these events, bacteria have evolved "DNA replication restart" mechanisms to reload replisomes onto abandoned replication forks. The Escherichia coli PriA DNA helicase orchestrates this process by recognizing and remodeling replication forks and recruiting additional proteins that help to drive replisome reloading. We have identified a conserved sequence motif within a linker region of PriA that docks into a groove on the exterior of the PriA helicase domain. Alterations to the motif reduce the apparent processivity and attenuate structure-specific helicase activity in PriA, implicating the motif as a potential autoregulatory element in replication fork processing. The study also suggests that multiple PriA molecules may function in tandem to enhance DNA unwinding processivity, highlighting an unexpected similarity between PriA and other DNA helicases. [ABSTRACT FROM AUTHOR]

Objective: The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin what results in melting of a double-stranded DNA (dsDNA) and formation of single-stranded DNA (ssDNA) region where the replication machinery can assemble. For prokaryotic initiators it was shown that they interact with the formed ssDNA and that this interaction is required for the replication activity. The ability to interact with ssDNA was also shown for Saccharomyces cerevisiae replication initiation protein complex ORC. For Archaea, which combine features of both prokaryotic and eukaryotic organisms, there was no evidence whether DNA replication initiators can interact with ssDNA. We address this issue in this study. Results: Using purified Orc1 protein from Aeropyrum pernix (ApOrc1) we analyzed its ability to interact with ssDNA containing sequence of an AT-rich region of the A. pernix origin Ori1 as well as with homopolymers of thymidine (polyT) and adenosine (polyA). The Bio-layer interferometry, surface plasmon resonance and microscale thermophoresis showed that the ApOrc1 can interact with ssDNA and it binds preferentially to T-rich ssDNA. The hydrolysis of ATP is not required for this interaction. [ABSTRACT FROM AUTHOR]

We analyse the effects of the environment (solvent quality, presence of extended structures -- crowded environment) that may have impact on the order of the transition between denaturated and bounded DNA states and lead to changes in the scaling laws that govern conformational properties of DNA strands. We find that the effects studied significantly influence the strength of the first order transition. To this end, we re-consider the Poland-Scheraga model and apply a polymer field theory to calculate entropic exponents associated with the denaturated loop distribution. For the d = 3 case, the corresponding diverging ε = 4 - d expansions are evaluated by restoring their convergence via the resummation technique. For the space dimension d = 2, the exponents are deduced from mapping the polymer model onto a two-dimensional random lattice, i.e., in the presence of quantum gravity. We also show that the first order transition is further strengthened by the presence of extended impenetrable regions in a solvent that restrict the number of the macromolecule configurations. [ABSTRACT FROM AUTHOR]

RESEARCH ARTICLE| JULY 16 2021 Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site Víctor Juarez-Quintero; Antolín Peralta-Castro; Claudia G. Benítez Cardoza; Tom Ellenberger; Luis G. Brieba Crossmark: Check for Updates Biochem J (2021) 478 (13): 2665–2679. https://doi.org/10.1042/BCJ20200922 Article history Share Icon Share Cite Icon Cite Get Permissions The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended β-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended β-hairpin is involved in nucleotide incorporation on substrates with 5′-overhangs longer than 2 nt, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3′–5′ exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating β-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels. [ABSTRACT FROM AUTHOR]

The Escherichia coli UvrD protein is a superfamily 1, non-hexameric DNA helicase that plays a crucial role in repair mechanisms. Previous studies suggested that wild-type UvrD has optimal activity in its oligomeric form. Nevertheless, a conflicting monomer model was proposed using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). Here, single-molecule direct visualization of UvrDΔ40C revealed that two or three UvrDΔ40C molecules were simultaneously involved in DNA unwinding, presumably in an oligomeric form, similar to that with wild-type UvrD. Thus, single-molecule direct visualization of nucleic acid-binding proteins provides quantitative and kinetic information to address their fundamental mechanisms. [ABSTRACT FROM AUTHOR]

• Difficulties of obtaining DNA for human identification in forensic cases. • Effects of incineration on bone and subsequent DNA yield. • The advantages and disadvantages of demineralisation prior to DNA extraction. • Compare various DNA extraction protocols (chelex, phenol/chloroform, solid-phase). • Explore and discuss areas for future research. Identification of incinerated human remains may rely on genetic analysis of burned bone which can prove far more challenging than fresh tissues. Severe thermal insult results in the destruction or denaturation of DNA in soft tissues, however genetic material may be preserved in the skeletal tissues. Considerations for DNA retrieval from these samples include low levels of exogenous DNA, the dense, mineralised nature of bone, and the presence of contamination, and qPCR inhibitors. This review collates current knowledge in three areas relating to optimising DNA recovery from burned bone: 1) impact of burning on bone and subsequent effects on sample collection, 2) difficulties of preparing burned samples for DNA extraction, and 3) protocols for bone decalcification and DNA extraction. Bone decalcification and various DNA extraction protocols have been tested and optimised for ancient bone, suggesting that prolonged EDTA (Ethylenediaminetetraacetic acid) demineralisation followed by solid-phased silica-based extraction techniques provide the greatest DNA yield. However, there is significantly less literature exploring the optimal protocol for incinerated bones. Although burned bone, like ancient and diagenetic bone, can be considered "low-copy", the taphonomic processes occurring are likely different. As techniques developed for ancient samples are tailored to deal with bone that has been altered in a particular way, it is important to understand if burned bone undergoes similar or different changes. Currently the effects of burning on bone and the DNA within it is not fully understood. Future research should focus on increasing our understanding of the effects of heat on bone and on comparing the outcome of various DNA extraction protocols for these tissues. [ABSTRACT FROM AUTHOR]

The issue of the nucleation and slow closure mechanisms of non-superhelical stress-induced denatur-ation bubbles in DNA is tackled using coarse-grained MetaDynamics and Brownian simulations. A minimal mesoscopic model is used where the double helix is made of two interacting bead-spring rotating strands with a prescribed torsional modulus in the duplex state. We demonstrate that timescales for the nucleation (respectively, closure) of an approximately 10 base-pair bubble, in agreement with experiments, are associated with the crossing of a free-energy barrier of 22 kBT (respectively, 13 kBT) at room temperature T. MetaDynamics allows us to reconstruct accurately the free-energy landscape, to show that the free-energy barriers come from the difference in torsional energy between the bubble and duplex states, and thus to highlight the limiting step, a collective twisting, that controls the nucleation/closure mechanism, and to access opening time scales on the millisecond range. Contrary to small breathing bubbles, those more than 4 base-pair bubbles are of biological relevance, for example, when a pre-existing state of denaturation is required by specific DNA-binding proteins. [ABSTRACT FROM AUTHOR]

A deep understanding to the denaturation and renaturation behaviors of DNA in a confined state is fundamentally important to control the self-assembly of DNA in a chamber or channel for various applications. In this report, we study the denaturation and renaturation behaviors of short DNA confined in cylindrical and spherical spaces with the 3-Site-Per-Nucleotide coarse-grained DNA model applying the replica exchange molecular dynamics technology. It is found that as the confinement size decreases, the melting temperature Tm increases and the transition becomes broad. The analysis of the potential of mean force shows that the confinement increases the relative free energy of the denatured state of DNA and decreases the renaturation energy barrier. Besides the denatured and native states, the metastable parallel-stranded structure is also found. The simulation results show that the shapes of the confinement spaces and the short DNA sequences remarkably affect the renaturation behavior. In the cylindrical space, the DNA renaturation changes from randombinding to slithering-binding with the size of the confinement space decreasing. In contrast, the DNA renaturation in the spherical and symmetrical confinement space proceeds through strand binding and rolling. The relationship between the melting temperature and the confinement size, △Tm/Tm ~ Rc-v, is estimated and the exponential index v equals about 1.32 and 1.75 in the cylindrical and spherical confinements, respectively. It is further compared with the theoretical result of the rigid rod model and a qualitative agreement with the simulation is achieved. [ABSTRACT FROM AUTHOR]

In many bacteria and eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+(ATPases Associated with various cellular Activities) ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of Escherichia coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We have identified a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate that elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as in polymerase clamp loading and certain classes of DNA transposases. [ABSTRACT FROM AUTHOR]

Copy number variants (CNVs) of breast and/or ovarian cancer susceptibility genes are a known class of clinically significant variants.[1],[2] Recent advances in next-generation sequencing (NGS) methods and bioinformatics lead to cost-effective and unified workflow to detect CNV and short sequence variants simultaneously for a panel of genes.[3],[4] However, CNV detection resolution by routine gene panel NGS is often limited to individual exon level without exact CNV breakpoint and direction. Molecular-barcode NGS testing of the proband blood DNA for six genes ( I BRCA1 i , I BRCA2 i , I TP53 i , I PTEN i , I PALB2 i , and I CDH1 i ) detected a CNV of I PALB2 i exon 13 (the last coding exon of I PALB2 i , reference sequence NM 024675.3) at a copy number ratio of 1.57, which was interpreted as a germline heterozygous duplication. We applied the same reverse transcriptase PCR assay from Yang et al[7] on our proband blood RNA and showed that there is no abnormal splicing of I PALB2 i exon 13. [Extracted from the article]

Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool. Here, structures of the Cas12i1 R-loop complexes before and after target DNA cleavage and biochemical assays offer detailed insights into the mechanisms of target DNA duplex unwinding, R-loop formation and DNA cleavage, and suggest that Cas12i1 forms a 19-bp long DNA-RNA heteroduplex with the crRNA guide region. [ABSTRACT FROM AUTHOR]

The versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4–Rad23-Rad33 (yeast homologue of XPC–RAD23B–CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9–9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification. The conserved eukaryotic nucleotide excision repair (NER) pathway protects the genome from a wide variety of environmentally induced DNA lesions. Here, the authors provide insights into how NER is initiated on lesions by determining the cryo-EM structure of the yeast TFIIH/Rad4–Rad23-Rad33 complex bound to a DNA containing a single carcinogen-DNA adduct. [ABSTRACT FROM AUTHOR]

The underlying constraint of ultrashort pulsed laser ablation in both the clinical and micromachining setting is the uncertainty regarding the impact on the composition of material surrounding the ablated region. A heat model representing the laser-tissue interaction was implemented into a finite element suite to assess the cumulative temperature response of bone during ultrashort pulsed laser ablation. As an example, we focus on the extraction of mineralised collagen fibre micropillars. Laser induced heating can cause denaturation of the collagen, resulting in ultrastructural loss which could affect mechanical testing results. Laser parameters were taken from a used micropillar extraction protocol. The laser scanning pattern consisted of 4085 pulses, with a final radial pass being 22 μ m away from the micropillar. The micropillar temperature was elevated to 70.58 ∘ C , remaining 79.42 ∘ C lower than that of which we interpret as an onset for denaturation. We verified the results by means of Raman microscopy and Energy Dispersive X-ray Microanalysis and found the laser-material interaction had no effect on the collagen molecules or mineral nanocrystals that constitute the micropillars. We, thus, show that ultrashort pulsed laser ablation is a safe and viable tool to fabricate bone specimens for mechanical testing at the micro- and nanoscale and we provide a computational model to efficiently assess this. [ABSTRACT FROM AUTHOR]

Repetitive DNAs comprise large portion of eukaryote genomes. In genome projects, the assembly of repetitive DNAs is challenging due to the similarity between repeats, which generate ambiguities for alignment. Fluorescence in situ hybridization (FISH) is a powerful technique for the physical mapping of various sequences on chromosomes. This technique is thus very helpful in chromosome-based genome assemblies, providing information on the fine architecture of genomes and their evolution. However, various protocols are currently used for FISH mapping, most of which are relatively laborious and expensive, or work properly only with a specific type of probes or sequences, and there is a need for a universal and affordable FISH protocol. Here we tested a FISH protocol for mapping of different DNA repeats, such as multigene families (rDNAs, U snDNAs, histone genes), satellite DNAs, microsatellites, transposable elements, DOP-PCR products, and telomeric motif (TTAGG)n, on the chromosomes of various insects and other arthropods. Different cell types and stages obtained from diverse tissues were used. The FISH procedure proved high quality and reliable results in all experiments performed. We obtained data on the chromosomal distribution of DNA repeats in representatives of insects and other arthropods. Thus, our results allow us to conclude that the protocol is universal and requires only time adjustment for chromosome/DNA denaturation. The use of this FISH protocol will facilitate studies focused on understanding the evolution and role of repetitive DNA in arthropod genomes. [ABSTRACT FROM AUTHOR]

The involvement of non-canonical DNA structures, such as Gquadruplex (G4) DNA, in cancer development and progression has set the pace towards the renaissance of DNA-binding metal complexes. In this work, we report the DNA-binding of three Ni(II), Cu(II), Zn(II) complexes of a salphen-like N4-donor ligand, bearing two imidazole groups condensed with a phenylenediamine moiety. Both duplex and G4 DNAs derived from human telomeres (hTelo), and a sequence mimicking the promoter of the oncogene myc (c-myc) were studied. UV-Vis and circular dichroism spectroscopic binding studies pointed out that, while all the three complexes bind the selected oligonucleotides, the Cu(II) derivative is the strongest and G4-selective compound of the series. Lastly, FRET DNA melting assay results on the Cu(II) complex/hTelo G4 system were interpreted by a loop-binding mechanism of interaction, as corroborated by molecular dynamics (MD) simulations. [ABSTRACT FROM AUTHOR]

[Display omitted] Theranostic nanoprobes can potentially integrate imaging and therapeutic capabilities into a single platform, offering a new personalized cancer diagnostic tool. However, there is a growing concern that their clinical application is not safe, particularly due to metal-containing elements, such as the gadolinium used in magnetic resonance imaging (MRI). We demonstrate for the first time that the photothermal melting of the DNA duplex helix was a reliable and versatile strategy that enables the on-demand degradation of the gadolinium-containing MRI reporter gene from polydopamine (PDA)-based theranostic nanoprobes. The combination of chemotherapy (doxorubicin) and photothermal therapy, which leads to the enhanced anti-tumor effect. In vivo MRI tracking reveals that renal filtration was able to rapidly clear the free gadolinium-containing MRI reporter from the mice body. This results in a decrease in the long-term toxic effect of theranostic MRI nanoprobes. Our findings may pave the way to address toxicity issues of the theranostic nanoprobes. [ABSTRACT FROM AUTHOR]

The BRCA2 tumor suppressor is a DNA double‐strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2‐deficient cells spontaneously accumulate DNA‐RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD‐box RNA helicase DDX5 as a BRCA2‐interacting protein. DDX5 associates with DNA‐RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA‐RNA hybrid‐unwinding activity of DDX5 helicase. An impaired BRCA2‐DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2‐T207A, reduces the association of DDX5 with DNA‐RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA‐RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal. SYNOPSIS: Cells lacking the BRCA2 tumor suppressor spontaneously accumulate DNA‐RNA hybrids, but the specific BRCA2 role in their suppression has remained unclear. Here, RNA helicase DDX5 is found to interact with BRCA2 and participate in R‐loop ressolution to allow homologous recombination (HR) repair. BRCA2 and DDX5 cooperate to unwind DNA double‐strand break‐associated R‐loops at transcribed regions.BRCA2 supports DDX5 association with DNA double‐strand breaks.Interaction between BRCA2 and DDX5 facilitates timely repair of DNA damage by HR.The breast‐cancer‐associated T207A mutation alters BRCA2 interaction with DDX5 and slows the kinetics of HR repair. [ABSTRACT FROM AUTHOR]

In mammalian cells, the bulky DNA adducts caused by ultraviolet radiation are mainly repaired via the nucleotide excision repair (NER) pathway; some defects in this pathway lead to a genetic disorder known as xeroderma pigmentosum (XP). Ribosomal protein S3 (rpS3), a constituent of the 40S ribosomal subunit, is a multi-functional protein with various extra-ribosomal functions, including a role in the cellular stress response and DNA repair-related activities. We report that rpS3 associates with transcription factor IIH (TFIIH) via an interaction with the xeroderma pigmentosum complementation group D (XPD) protein and complements its function in the NER pathway. For optimal repair of UV-induced duplex DNA lesions, the strong helicase activity of the TFIIH complex is required for unwinding damaged DNA around the lesion. Here, we show that XP-D cells overexpressing rpS3 showed markedly increased resistance to UV radiation through XPD and rpS3 interaction. Additionally, the knockdown of rpS3 caused reduced NER efficiency in HeLa cells and the overexpression of rpS3 partially restored helicase activity of the TFIIH complex of XP-D cells in vitro. We also present data suggesting that rpS3 is involved in post-excision processing in NER, assisting TFIIH in expediting the repair process by increasing its turnover rate when DNA is damaged. We propose that rpS3 is an accessory protein of the NER pathway and its recruitment to the repair machinery augments repair efficiency upon UV damage by enhancing XPD helicase function and increasing its turnover rate. [ABSTRACT FROM AUTHOR]

Background and Aim: Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance caused by homozygous SMN1 deletions. Although SMA has been considered as incurable, newly developed drugs improve life prognoses and motor functions of patients. To maximize the efficacy of the drugs, SMA patients should be treated before symptoms become apparent. Thus, newborn screening for SMA is strongly recommended. In this study, we aim to establish a new simple screening system based on DNA melting peak analysis. Materials and Methods: A total of 124 dried blood spot (DBS) on FTA® ELUTE cards (51 SMN1-deleted patients with SMA, 20 carriers, and 53 controls) were punched and subjected to direct amplification of SMN1 and CFTR (reference gene). Melting peak analyses were performed to detect SMN1 deletions from DBS samples. Results: A combination of allele-specific polymerase chain reaction (PCR) and melting peak analyses clearly distinguished the DBS samples with and without SMN1. Compared with the results of fresh blood samples, our new system yielded 100% sensitivity and specificity. The advantages of our system include (1) biosafe collection, transfer, and storage for DBS samples, (2) obviating the need for DNA extraction from DBS preventing contamination, (3) preclusion of fluorescent probes leading to low PCR cost, and (4) fast and high-throughput screening for SMN1 deletions. Conclusion: We demonstrate that our system would be applicable to a real-world newborn screening program for SMA, because our new technology is efficient for use in routine clinical laboratories that do not have highly advanced PCR instruments. [ABSTRACT FROM AUTHOR]

Fluoroquinolones are excessively prescribed in the therapy of genital tract infections. However, fluoroquinolone treatment may be associated with fertility problems, insufficient information concerning their effect on fertility are present. The goal of this study is to elucidate the effects of two fluoroquinolones treatment i.e. ciprofloxacin and levofloxacin on certain sperm function parameters and even on reproductive tissues and to investigate whether these two antibiotics can affect sperm DNA integrity or chromatin quality. Eighty mature Albino male rats adult were enrolled in this study. The animals were randomly divided into 10 groups (8 animals in each). Four groups treated with levofloxacin with a dose of 37.5 mg/kg/day and 75 mg/kg/day of levofloxacin and four ciprofloxacin treated groups which were treated with a dose of 40mg/kg/day and 80 mg/kg/ day of ciprofloxacin and two control groups, for each treatment dose the treatment continue for either 14 days or 28 days. Certain epididymal sperm function parameters namely : sperm concentration, sperm motility and morphologically normal sperm percentage were analyzed. In addition to analysis of sperm DNA integrity and chromatin quality, histopathology of testes and epididymus were examined. The results shown that certain sperm function parameters were not significantly affected when levofloxacin and ciprofloxacin were administered orally for 14 days. While, a significant reduction in; sperm concentration, percentage of morphologically normal sperm and sperm motility were observed when the two medicines were administered for 28 days. A significant increase in the level of DNA fragmentation with a significant reduction in chromatin quality were also observed in fluoroquinolone treated groups following 28 days treatment. The testes of rat treated with fluoroquinolones showed changes in the number of spermatozoa in some seminiferous and epididymal tubules when the two medicine were applied in high doses and for 28 days. These results indicate that fluoroquinolone treatment can be used for 14 days in rats (which equal 28 days in men) with no harmful effects on certain sperm parameters and adversely affect fertility potential in male in a dose and time dependent manner mainly by interfering with sperm DNA integrity level [ABSTRACT FROM AUTHOR]

Soon after its introduction in 1987, polymerase chain reaction (PCR) has become a technique widely employed in diagnostic medical devices and forensic science with the intention of amplifying genetic information. PCR prescribes that each of its cycles must include a heating subprocess at 95 °C or more (denominated DNA denaturation and provided for allowing a claimed orderly separation of the two complementary nucleotides strands), which can produce significant damage to DNA, caused by high-speed collisions with surrounding molecules. Since such disruption should be prevented in order to reliably employ PCR, a study of the mechanics of such loss of structural integrity is herein presented, preceded by a review of the fundamental literature which has elucidated the effects of molecular agitation on DNA fragmentation. The main conclusion of this retrospective survey is that the body of examined theoretical and experimental evidence consistently and redundantly confirms scarce resilience and significant loss of structural integrity when DNA is heated at temperatures above 90 °C, even for 1 minute. Such conclusion contradicts the claimed paradigm of PCR fidelity and raises the concern that, at least for long sequences, if PCR can amplify some information, such amplified information may be unreliable for diagnostic or forensic applications, since it originates from sequences of nucleotides subjected to random fragmentation and reaggregation. Such a low-reliability scenario should be preventively considered in the various fields where DNA amplification methodologies are employed which provide for high-temperature heating under conditions equal to or similar to those prescribed by the PCR protocols reviewed in this study.

Melamine binding to polythymine (poly-T) DNA has been widely used to develop biosensors for the detection of melamine. In this work, SYBR Green I (SGI) was used to stain the binding reaction, and using DNA melting experiments, it was confirmed that poly-T DNA formed intramolecular binding complexes to bind multiple melamine molecules. In addition, while this system was insensitive to ionic strength, longer DNA and lower pH favored the binding of melamine. A fluorescence resonance energy transfer (FRET) sensor was designed by labeling a TAMRA and a Cy5 fluorophore on the two ends of T30 DNA, respectively. In the presence of SGI, FRET-based ratiometric detection was achieved with an apparent Kd of 85 μM and a limit of detection of 10.7 μM melamine. Without SGI, the sensitivity of detection was decreased by 47-fold. Interference from Hg2+ can be masked by adding EDTA. Detection of melamine in milk was achieved with recovery rates from 87.0 to 101.7%. This study has provided both basic biochemical insights and a ratiometric sensor for highly sensitive and robust detection of melamine. [ABSTRACT FROM AUTHOR]

Using glucose oxidase and salmon testis-derived DNA molecules, we sought to extend the recently proposed idea of interfacial adsorption denaturation. The surface pressure-time (p-t) isotherm of the glucose oxidase Gibbs monolayer exhibited a rapid increase in surface pressure and a relatively prompt transition to a liquid condensed film. The appearance of this rapid liquid expansion phase occurred much earlier than that previously identified for lysozyme, trypsin, cytochrome C, and luciferase. This experimental finding was linked to the number of hydrophobic residues in the constituent unit, and the number of hydrophobic residues in glucose oxidase was the highest among these biomolecules. On the other hand, DNA molecules do not have such hydrophobic groups, or present a positive surface on the p-t curve. However, interfacial adsorption occurred, and the existence of molecules at the air/water interface was confirmed, even in the two-dimensional gas phase state. Furthermore, it was confirmed that an increase in surface pressure was detected during the formation of a mixed film of DNA molecules and biomolecules, forming a stable Gibbs monolayer. This mimic the behavior of mixed monolayer formation with matrix molecules in Langmuir monolayers. Moreover, it was clarified that the interfacial adsorption denaturation behavior changed when pH dependence was evaluated considering the isoelectric point of the biomolecular group. [ABSTRACT FROM AUTHOR]