Single-molecule characterization of SV40 replisome and novel factors: human FPC and Mcm10.

The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45-MCM-GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system.

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.

Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing.

DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks.

BACKGROUND: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. RESULTS: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. CONCLUSIONS: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

Severe Combined Immunodeficiency from a Homozygous DNA Ligase 1 Mutant with Reduced Catalytic Activity but Increased Ligation Fidelity.

A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.

Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2.

Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/µL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin-ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin-ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

Resolution of the Holliday junction recombination intermediate by human GEN1 at the single-molecule level.

Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ) after the dissolution of the nuclear membrane. GEN1 dimerization has been suggested to play key role in the resolution of the HJ, but the kinetic details of its reaction remained elusive. Here, single-molecule FRET shows how human GEN1 binds the HJ and always ensures its resolution within the lifetime of the GEN1-HJ complex. GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. GEN1 monomer remains tightly bound with no apparent dissociation until GEN1 dimer is formed and the HJ is fully resolved. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction. Furthermore, GEN1 monomer binds singly-cleaved HJ tighter than intact HJ providing a fail-safe mechanism if GEN1 dimer or one of its monomers dissociates after the first cleavage. The tight binding of GEN1 monomer to intact- and singly-cleaved HJ empowers it as the last resort to process HJs that escape the primary mechanisms.

Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway.

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

Transcriptomic and metabolomic changes in postharvest sugarbeet roots reveal widespread metabolic changes in storage and identify genes potentially responsible for respiratory sucrose loss.

Endogenous metabolism is primarily responsible for losses in sucrose content and processing quality in postharvest sugarbeet roots. The genes responsible for this metabolism and the transcriptional changes that regulate it, however, are largely unknown. To identify genes and metabolic pathways that participate in postharvest sugarbeet root metabolism and the transcriptional changes that contribute to their regulation, transcriptomic and metabolomic profiles were generated for sugarbeet roots at harvest and after 12, 40 and 120 d storage at 5 and 12°C and gene expression and metabolite concentration changes related to storage duration or temperature were identified. During storage, 8656 genes, or 34% of all expressed genes, and 225 metabolites, equivalent to 59% of detected metabolites, were altered in expression or concentration, indicating extensive transcriptional and metabolic changes in stored roots. These genes and metabolites contributed to a wide range of cellular and molecular functions, with carbohydrate metabolism being the function to which the greatest number of genes and metabolites classified. Because respiration has a central role in postharvest metabolism and is largely responsible for sucrose loss in sugarbeet roots, genes and metabolites involved in and correlated to respiration were identified. Seventy-five genes participating in respiration were differentially expressed during storage, including two bidirectional sugar transporter SWEET17 genes that highly correlated with respiration rate. Weighted gene co-expression network analysis identified 1896 additional genes that positively correlated with respiration rate and predicted a pyruvate kinase gene to be a central regulator or biomarker for respiration rate. Overall, these results reveal the extensive and diverse physiological and metabolic changes that occur in stored sugarbeet roots and identify genes with potential roles as regulators or biomarkers for respiratory sucrose loss.

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public's health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens.

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

An open-source, automated, and cost-effective platform for COVID-19 diagnosis and rapid portable genomic surveillance using nanopore sequencing.

The COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use-with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.

The transcriptional regulator LEUNIG_HOMOLOG regulates mucilage release from the Arabidopsis testa.

Exposure of the mature Arabidopsis (Arabidopsis thaliana) seed to water results in the rapid release of pectinaceous mucilage from the outer cells of the testa. Once released, mucilage completely envelops the seed in a gel-like capsule. The physical force required to rupture the outer cell wall of the testa comes from the swelling of the mucilage as it expands rapidly following hydration. In this study, we show that mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) cause a mucilage extrusion defect due to altered mucilage swelling. Based on sugar linkage and immunomicroscopic analyses, we show that the structure of luh mucilage is altered, having both an increase in substituted rhamnogalacturonan I and in methyl-esterified homogalacturonan. Also correlated with the structural modification of luh mucilage is a significant decrease in MUCILAGE MODIFIED2 (MUM2; a ß-galactosidase) expression in the luh seed coat, raising the possibility that reduced activity of this glycosidase is directly responsible for the luh mucilage defects. Consistent with this is the structural similarity between mum2 and luh mucilage as well as the observation that elevating MUM2 expression in luh mutants completely suppresses the mucilage extrusion defect. Suppression of the luh mutant phenotype was also observed when LEUNIG, a transcriptional corepressor closely related to LUH, was introduced in luh mutants under the control of the LUH promoter. Based on these data, we propose a new model for the regulation of pectin biosynthesis during plant growth and development.

QTL Mapping of Adult Plant Resistance to Stripe Rust in a Doubled Haploid Wheat Population.

Stripe rust caused by Puccinia striiformis Westend. f. sp. tritici. is a major bread wheat disease worldwide with yield losses of up to 100% under severe disease pressure. The deployment of resistant cultivars with adult plant resistance to the disease provides a long-term solution to stripe rust of wheat. An advanced line from the International Winter Wheat Improvement Program (IWWIP) 130675 (Avd/Vee#1//1-27-6275/Cf 1770/3/MV171-C-17466) showed a high level of adult plant resistance to stripe rust in the field. To identify the adult plant resistance genes in this elite line, a mapping population of 190 doubled haploid (DH) lines was developed from a cross between line 130675 and the universal stripe rust-susceptible variety Avocet S. The DH population was evaluated at precision wheat stripe rust phenotyping platform, in Izmir during 2019, 2020, and 2021 cropping seasons under artificial inoculations. Composite interval mapping (CIM) identified two stable QTLs QYr.rcrrc-3B.1, and QYr.rcrrc-3B.2, which were detected in multiple years. In addition to these two QTLs, five more QTLs, QYr.rcrrc-1B, QYr.rcrrc-2A, QYr.rcrrc-3A, QYr.rcrrc-5A, and QYr.rcrrc-7D, were identified, which were specific to the cropping year (environment). All QTLs were derived from the resistant parent, except QYr.rcrrc-3A. The significant QTLs explained 3.4-20.6% of the phenotypic variance. SNP markers flanking the QTL regions can be amenable to marker-assisted selection. The best DH lines with high yield, end-use quality, and stripe rust resistance can be used for further selection for improved germplasm. SNP markers flanking the QTL regions can aid in identifying such lines.

Exploring the Genetic Diversity and Population Structure of Wheat Landrace Population Conserved at ICARDA Genebank.

Landraces are considered a valuable source of potential genetic diversity that could be used in the selection process in any plant breeding program. Here, we assembled a population of 600 bread wheat landraces collected from eight different countries, conserved at the ICARDA's genebank, and evaluated the genetic diversity and the population structure of the landraces using single nucleotide polymorphism (SNP) markers. A total of 11,830 high-quality SNPs distributed across the genomes A (40.5%), B (45.9%), and D (13.6%) were used for the final analysis. The population structure analysis was evaluated using the model-based method (STRUCTURE) and distance-based methods [discriminant analysis of principal components (DAPC) and principal component analysis (PCA)]. The STRUCTURE method grouped the landraces into two major clusters, with the landraces from Syria and Turkey forming two clusters with high proportions of admixture, whereas the DAPC and PCA analysis grouped the population into three subpopulations mostly according to the geographical information of the landraces, i.e., Syria, Iran, and Turkey with admixture. The analysis of molecular variance revealed that the majority of the variation was due to genetic differences within the populations as compared with between subpopulations, and it was the same for both the cluster-based and distance-based methods. Genetic distance analysis was also studied to estimate the differences between the landraces from different countries, and it was observed that the maximum genetic distance (0.389) was between the landraces from Spain and Palestine, whereas the minimum genetic distance (0.013) was observed between the landraces from Syria and Turkey. It was concluded from the study that the model-based methods (DAPC and PCA) could dissect the population structure more precisely when compared with the STRUCTURE method. The population structure and genetic diversity analysis of the bread wheat landraces presented here highlight the complex genetic architecture of the landraces native to the Fertile Crescent region. The results of this study provide useful information for the genetic improvement of hexaploid wheat and facilitate the use of landraces in wheat breeding programs.

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics.

The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

Cryo-electron structures of the extreme thermostable enzymes Sulfur Oxygenase Reductase and Lumazine Synthase.

Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous environment. Here, we present high resolution cryo-EM structures of two thermostable enzymes that exhibit multimeric cage-like structures arranged into two different point-group symmetries. First, we determined the structure of the Sulfur Oxygenase Reductase (SOR) enzyme that catalyzes both the oxygenation and disproportionation of elemental sulfur in Archea and is composed of 24 homomeric units each of MW ≃ 35 kDa arranged in octahedral symmetry. The structure of SOR from Acidianus ambivalens (7X9W) was determined at 2.78 Å resolution. The active site of each subunit inside the central nanocompartment is composed of Fe3+ coordinated to two water molecules and the three amino acids (H86, H90 and E114). Second, we determined the structure of Lumazine Synthase (LS) from Aquifex aeolicus (7X7M) at 2.33 Å resolution. LS forms a cage-like structure consisting of 60 identical subunits each of MW ≃ 15 kDa arranged in a strict icosahedral symmetry. The LS subunits are interconnected by ion-pair network. Due to their thermostability and relatively easy purification scheme, both SOR and LS can serve as a model for the catalytic and structural characterization of biocatalysts as well as a benchmark for cryo-EM sample preparation, optimization of the acquisition parameters and 3D reconstruction.