Nuclease Activity Research Articles

Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11’s nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Corrected and republished from: "Vibrio fischeri Possesses Xds and Dns Nucleases That Differentially Influence Phosphate Scavenging, Aggregation, Competence, and Symbiotic Colonization of Squid".

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.

Detailed analysis of agro-industrial byproducts/wastes to enable efficient sorting for various agro-industrial applications

Agriculture-based industries generate huge amounts of byproducts/wastes every year, which are not exploited or disposed efficiently posing an environmental problem with implications to human and animal health. Finding strategies to increase the recycling of agro-industrial byproducts/wastes (AIBWs) is a primary objective of the current study. A thorough examination of AIBWs in conjunction with experimental research is proposed to facilitate sorting for various agro-industrial applications and consequently increasing byproduct/waste utilization. Accordingly, two sustainable, locally available sources of AIBWs, namely, wheat bran (WB) and garlic straw and peels (GSP) were studied in detail including content and composition of proteins, phytohormones and nutritional elements, as well as the effect of AIBW extracts on plant and microbial growth. Hundreds of proteins were recovered from AIBW mainly from WBs, including chaperons, metabolite and protein modifying enzymes, and antimicrobial proteins. In-gel assays showed that WB and GSP possess high protease and nuclease activities. Conspicuously, phytohormone analysis of AIBWs revealed the presence of high levels of strigolactones, stimulants of seed germination of root parasitic weeds, as well as indole acetic acid (IAA) and abscisic acid (ABA). Garlic straw extract strongly inhibited germination of the weed Amaranthus palmeri but not of Abutilon theophrasti and all examined AIBWs significantly affected post-germination growth. Bacterial growth was strongly inhibited by garlic straw, but enhanced by WBs, which can be used at least partly as a bacterial growth medium. Thus, an in-depth examination of AIBW characteristics will enable appropriate sorting for diverse agro-industrial applications, which will increase their utilization and consequently their economic value.

Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.2 and C11 subunits, respectively. In contrast, Pol II requires the transcription factor (TF)IIS to confer robust nuclease activity. We recently reported that in the absence of the A12.2 subunit Pol I reverses bond formation by pyrophosphorolysis in the absence of added PPi, indicating slow PPi release. Thus, we hypothesized that Pol II, naturally lacking TFIIS, would reverse bond formation through pyrophosphorolysis. Here we report the results of transient-state kinetic experiments to examine the addition of nine nucleotides to a growing RNA chain catalyzed by Pol II. Our results indicate that Pol II reverses bond formation by pyrophosphorolysis in the absence of added PPi. We propose that, in the absence of endonuclease activity, this bond reversal may represent kinetic proofreading. Thus, given the hypothesis that Pol I evolved from Pol II through the incorporation of general transcription factors, pyrophosphorolysis may represent a more ancient form of proofreading that has been evolutionarily replaced with nuclease activity.

Recent advances on the DNA interaction properties of Schiff bases and their metal complexes

Cancer has become the second leading reason for death in the world. Cell biology states that the bulk of anti‐cancer medications focuses on DNA. A study of how complexes of metals interact with DNA is crucial in creation of the effective chemotherapy drugs. The interactions of DNA with Schiff base and their metal complexes are a thrust area for many researchers, and many papers have reported on them. An amino molecule reacts with carbonyl group to form a variety of chemicals called Schiff bases. Many studies have shown that Schiff bases have compounds that affect biological processes. Due to their potential use as novel reagents for biotechnology and medicine, the research of interactions between transition metal complexes and nucleic acids has grown significantly during the past few decades. It was shown that several metal Schiff base complexes may intercalate DNA. Small molecules can attach themselves to DNA through different ways, such as intercalation and groove binding. The main method to measure nuclease activity was gel electrophoresis, and the ligand alone had much lower activity than the complex. To set the stage for this analysis, a brief overview of Schiff bases and their complexes is provided. Further, this review provides an overview of the DNA interactions brought forth by Schiff base metal complexes. It also draws attention to new developments in the study of Schiff bases and their metal complexes and their prospective use as a bioactive core for DNA cleavage and binding.

PYOCIN S5 ACTIVITY AGAINST PSEUDOMONAS SYRINGAE PHYTOPATHOGENIC STRAINS

Pseudomonas syringae is the most frequently isolated bacterial plant pathogens. Previously we revealed a number of Pseudomonas aeruginosa strains – producers of bacteriocins (pyocins) with high activity against P. syringae. However, it was unknown, which type of pyocins possesses the activity against P. syringae strains. S5 and S1 pyocin genes were observed in the genome of most active strain–producer P. aeruginosa UCM В-333. Pyocins were induced by nalidixic acid (100 μg/ml). Using DEAE-cellulose chromatography we isolated two bacteriocin groups from initial lysate. Substances from the 10th fraction had nuclease activity (pyocin S1) and the 27th fraction - pore-forming activity (pyocin S5). It was revealed that S1 pyocin didn’t lead to growth inhibition of studied bacterial plant pathogens. However pyocin S5 caused appearance of growth inhibition area with diameter 14-20 mm. So, pyocins of S1 and S5 types are active substances in P. aeruginosa UCM В-333 lysates, but only S5 pyocin possess activity against P. syringae strains. fpt A receptor gene was revealed in genomes of all analyzed cultures, including used phytopathogenic strains. This fact can explain wide spectrum of pyocin S5 activity. It is suggested that pyocin S5 can be a potential biocontrol agent against phytopathogenic P. syringae strains.

DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

Posttranscriptional regulation of FAN1 by miR-124-3p at rs3512 underlies onset-delaying genetic modification in Huntington’s disease

Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.

Decrypting Molecular Mechanisms Involved in Counteracting Copper and Nickel Toxicity in Jack Pine (Pinus banksiana) Based on Transcriptomic Analysis.

The remediation of copper and nickel-afflicted sites is challenged by the different physiological effects imposed by each metal on a given plant system. Pinus banksiana is resilient against copper and nickel, providing an opportunity to build a valuable resource to investigate the responding gene expression toward each metal. The objectives of this study were to (1) extend the analysis of the Pinus banksiana transcriptome exposed to nickel and copper, (2) assess the differential gene expression in nickel-resistant compared to copper-resistant genotypes, and (3) identify mechanisms specific to each metal. The Illumina platform was used to sequence RNA that was extracted from seedlings treated with each of the metals. There were 449 differentially expressed genes (DEGs) between copper-resistant genotypes (RGs) and nickel-resistant genotypes (RGs) at a high stringency cut-off, indicating a distinct pattern of gene expression toward each metal. For biological processes, 19.8% of DEGs were associated with the DNA metabolic process, followed by the response to stress (13.15%) and the response to chemicals (8.59%). For metabolic function, 27.9% of DEGs were associated with nuclease activity, followed by nucleotide binding (27.64%) and kinase activity (10.16%). Overall, 21.49% of DEGs were localized to the plasma membrane, followed by the cytosol (16.26%) and chloroplast (12.43%). Annotation of the top upregulated genes in copper RG compared to nickel RG identified genes and mechanisms that were specific to copper and not to nickel. NtPDR, AtHIPP10, and YSL1 were identified as genes associated with copper resistance. Various genes related to cell wall metabolism were identified, and they included genes encoding for HCT, CslE6, MPG, and polygalacturonase. Annotation of the top downregulated genes in copper RG compared to nickel RG revealed genes and mechanisms that were specific to nickel and not copper. Various regulatory and signaling-related genes associated with the stress response were identified. They included UGT, TIFY, ACC, dirigent protein, peroxidase, and glyoxyalase I. Additional research is needed to determine the specific functions of signaling and stress response mechanisms in nickel-resistant plants.

A responsive disulfide bond switch aptamer prodrug exhibiting enhanced stability and anticancer efficacy

Aptamers have shown significant potential in treating diverse diseases. However, challenges such as stability and drug delivery limited their clinical application. In this paper, the development of AS1411 prodrug-type aptamers for tumor treatment was introduced. A Short oligonucleotide was introduced at the end of the AS1411 sequence with a disulfide bond as responsive switch. The results indicated that the aptamer prodrugs not only enhanced the stability of the aptamer against nuclease activity but also facilitated binding to serum albumin. Furthermore, in the reducing microenvironment of tumor cells, disulfide bonds triggered drug release, resulting in superior therapeutic effects in vitro and in vivo compared to original drugs. This paper proposes a novel approach for optimizing the structure of nucleic acid drugs, that promises to protect other oligonucleotides or secondary structures, thus opening up new possibilities for nucleic acid drug design.

Biofouling mitigation of Nb2AlC and Mo3AlC2 MXene-precursors doped polyether sulfone mixed matrix membranes for pathogen microorganisms

This study explores the incorporation of Nb2AlC and Mo3AlC2 MAX phases, known for their nano-layered structure, into polyether sulfone (PES) membranes to enhance their antifouling and permeability properties for pathogen microorganism filtration against bovine serum albumin (BSA) and Escherichia coli (E. coli). The composite membranes were characterized for their structural and morphological properties, and their performance in mitigating biofouling was evaluated. The structural characterizations have been performed for all the prepared MAX phases and corresponding composite membranes. The antioxidant ability of Nb2AlC and Mo3AlC2 MAX phases was defined by the DPPH radical scavenging assay, and the highest antioxidant ability was found to be 59.35 %, while 53.69 % scavenging potential was recorded for 16 at 100 mg/L. The percentage scavenging ability was raised with an increase in concentrations. The antimicrobial properties of MAX phases, evaluated as the minimum inhibitory concentration, were stated against several pathogen microorganisms. The tested compounds of Nb2AlC and Mo3AlC2 composites containing MAX phases exhibited excellent chemical nuclease activity, and it was determined that Nb2AlC caused double strand DNA cleavage activity while Mo3AlC2 induced the complete fragmentation of the DNA molecule. Biofilm inhibition of Nb2AlC and Mo3AlC2 MAX phases was studied against Staphylococcus aureus, and Pseudomonas aeruginosa and the maximum biofilm inhibition of Nb2AlC and Mo3AlC2 MAX phases was found to be 77.15 % and 69.07 % against S. aureus and also 69.74 % and 65.01 % against P. aeruginosa. Furthermore, The Nb2AlC and Mo3AlC2 MAX phases demonstrated excellent E. coli growth inhibition of 100 % at 125 and 250 mg/L.

The Syntheses of Chromium Aluminum Carbide (Cr2AlC) MAXPhase and Cr2CTx MXene and Investigation of Their Antimicrobial Properties.

Chromium aluminum carbide (Cr2AlC) MAX phase and Cr2CTx (MXene-Cr) were synthesized by the pressureless sintering method and hydrothermal method, respectively. In addition to this, the free radical scavenging activities (FRSA) of MAX-Cr phase and MXene-Cr compounds were tested and compared with ascorbic acid and trolox as standard compounds. The obtained FRSA results of MAX-Cr phase and MXene-Cr were 42.82 and 59.64%, respectively, at 100mg/L concentration. MXene-Cr showed a 66.90% inhibitory effect on α-amylase at 200mg/L. The DNA nuclease activity of compounds was determined to be extremely satisfactory at 50, 100, and 200mg/L concentrations. Moreover, the prepared MAX-Cr phase and MXene-Cr were investigated for antimicrobial activity against six bacterial and two fungal strains by the broth microdilution method. Compounds provided more significant inhibition against Gram-positive bacteria than Gram-negative bacteria and fungi. MAX-Cr phase and MXene-Cr almost completely inhibited microbial cell viability at a 25mg/L concentration. Additionally, MXene-Cr showed 89.86% and 87.01% antibiofilm activity against S. aureus and P. aeruginosa, respectively, while the antibiofilm activity of the MAX-Cr phase was over 90%.

SARS-CoV2 Nsp1 is a metal-dependent DNA and RNA endonuclease.

Over recent years, we have been living under a pandemic, caused by the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). One of the major virulence factors of Coronaviruses is the Non-structural protein 1 (Nsp1), known to suppress the host cells protein translation machinery, allowing the virus to produce its own proteins, propagate and invade new cells. To unveil the molecular mechanisms of SARS-CoV2 Nsp1, we have addressed its biochemical and biophysical properties in the presence of calcium, magnesium and manganese. Our findings indicate that the protein in solution is a monomer and binds to both manganese and calcium, with high affinity. Surprisingly, our results show that SARS-CoV2 Nsp1 alone displays metal-dependent endonucleolytic activity towards both RNA and DNA, regardless of the presence of host ribosome. These results show Nsp1 as new nuclease within the coronavirus family. Furthermore, the Nsp1 double variant R124A/K125A presents no nuclease activity for RNA, although it retains activity for DNA, suggesting distinct binding sites for DNA and RNA. Thus, we present for the first time, evidence that the activities of Nsp1 are modulated by the presence of different metals, which are proposed to play an important role during viral infection. This research contributes significantly to our understanding of the mechanisms of action of Coronaviruses.

Enhanced editing efficiency in Arabidopsis with a LbCas12a variant harboring D156R and E795L mutations

AbstractCas12a (Cpf1), a Class 2 Type V CRISPR/Cas nuclease, has several unique attributes for genome editing and may provide a valuable alternative to Cas9. However, a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad application. In this report, we generated two variants, ttAsCas12 Ultra and ttLbCas12a Ultra harboring three (E174R, M537R, and F870L) or two (D156R and E795L) mutations, respectively, by combining the mutations from the temperature-tolerant variants ttAsCas12a (E174R) and ttLbCas12a (D156R), and those from the highly active variants AsCas12a Ultra (M537R and F870L) and LbCas12a Ultra (E795L). We compared editing efficiencies of the five resulting Cas12a variants (LbCas12a, ttLbCas12a, ttLbCas12a Ultra, AsCas12a Ultra, and ttAsCas12 Ultra) at six target sites of four genes in Arabidopsis (Arabidopsis thaliana). The variant ttLbCas12a Ultra, harboring the D156R and E795L mutations, exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22 °C. In addition, optimization of ttLbCas12a Ultra, by varying nuclear localization signal sequences and codon usage, further greatly improved editing efficiency. Collectively, our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis.

Photoaquation of cis-[Rh(dppz)(phen)Cl2]Cl complex prospective as potential light-activated anti-cancer agent

The photoactivated drug usage in anti-cancer therapy, the working mechanism of which doesn’t rely on the formation of reactive oxygen species, allows reducing damage to healthy tissue by controlling toxicity. For instance, light-activated Rh(III) complexes show nuclease activity after irradiation with UV and visible light. However, literature analysis shows a lack of descriptions of basic photochemical mechanisms. Here we report the study of cis-[Rh(dppz)(phen)Cl2]Cl (DPPZPHEN, dppz is dipyrido[3,2-a:2′,3′-c]phenazine, phen is 1,10-phenanthroline) photochemistry in water solutions using stationary photolysis, nanosecond laser flash photolysis and ultrafast pump–probe spectroscopy. After the irradiation with 308 nm light, DPPZPHEN undergoes aquation, which results in the substitution of one chloride ligand by a water molecule and leads to the formation of [Rh(dppz)(phen)(H2O)Cl]Cl2. UV–vis spectra of complex and capillary electrophoresis data were collected and discussed. Laser flash photolysis (355 nm) demonstrates intermediate absorption attributed to the lowest electronic excited state (triplet) of the initial complex. The study of primary photophysical processes showed a rather complicated behavior of transient absorption, which could be described by a set of four exponential functions. The early photophysical processes were described by the scheme including the formation of two excited states of different natures, namely a dppz-based IL state and an LMCT state.

Eukaryotic-driven directed evolution of Cas9 nucleases

BackgroundFurther advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity.ResultsHere, we develop a Eukaryotic Platform to Improve Cas Activity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity.ConclusionsWe report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.

A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.

Effect of Cu(II) compound containing dipicolinic acid on DNA damage: a study of antiproliferative activity and DNA interaction properties by spectroscopic, molecular docking and molecular dynamics approaches

A polymeric compound formulized as [Cu(µ-dipic)2{Na2(µ-H2O)4]n.2nH2O (I), where dipic is 2,6-pyridine dicarboxylic acid (dipicolinic acid, H2dipic), was synthesized by sonochemical irradiation. The initial in-vitro cytotoxic activity of this complex compared with renowned anticancer drugs like cisplatin, versus HCT116 colon cell lines, shows promising results. This study investigated the interaction mode between compound (I) and calf-thymus DNA utilizing a range of analytical techniques including spectrophotometry, fluorimetry, partition coefficient analysis, viscometry, gel electrophoresis and molecular docking technique. The results obtained from experimental methods reveal complex (I) could bind to CT-DNA via hydrogen bonding and van der Waals forces and the theoretical methods support it. Also, complex (I) indicates nuclease activity in the attendance of H2O2 and can act as an artificial nuclease to cleave DNA with high efficiency. Communicated by Ramaswamy H. Sarma

Investigation of advanced biological properties of carbon, carbon‐boron quantum dots, and copper (II) phthalocyanine nanoconjugates

Non‐peripherally, glycerol terminal groups substituted copper (II) phthalocyanine were non‐covalently (electrostatic and/or π–π interaction) attached to carbon (CQD) and carbon‐boron quantum dots (CBQD) to form QDs‐Pc nanoconjugate systems. Synthesized novel phthalocyanine compounds and QDs‐Pc conjugate systems were characterized using different spectroscopic techniques. Various biological assessments were applied to newly synthesized compounds. Conjugates 4 and 5 had a maximal free radical scavenging activity of 71.3% and 68.1% at a 100 mg/L concentration. Compounds exhibited high antidiabetic activities at 200 mg/L. Also, compounds showed significant DNA nuclease activity at all tested concentrations. The most efficient MIC value was obtained as 4 mg/L against Enterococcus hirae and Enterococcus feacalis. This MIC value was further decreased after photodynamic therapy, and it was observed that the antimicrobial effects of the compounds increased. Inhibition of microbial cell viability was obtained as 100% for all compounds. In addition, compounds exerted perfect biofilm inhibitory effects.