Bacterial Nucleases Research Articles

D-Methionine-induced DNases disperse established Burkholderia pseudomallei biofilms and promotes ceftazidime susceptibility

Burkholderia pseudomallei biofilm is correlated with pathogenesis, antibiotic resistance, and relapsing cases of melioidosis, leading to challenges in clinical management. There is increasing interest in employing biofilm dispersal agents as adjunctive treatments for biofilm-associated infections. Methionine (Met) has shown promise as an anti-biofilm agent by inducing bacterial DNase production, resulting in the degradation of extracellular DNA (eDNA) and dispersion of bacterial biofilm. In this study, we investigated the impact of 0.05–50 μM D-Met and L-Met on the 24-h established biofilm of a clinical isolate, B. pseudomallei H777. Our findings revealed the ability of D-Met and L-Met to disperse the established biofilm in a non-dose-dependent manner accompanied by eDNA depletion. Real-time PCR analysis further identified an up-regulation of bacterial nuclease genes, including recJ, eddB, nth, xth, and recD, in the presence of 0.05 μM D-Met. Similarly, recJ and eddB in B. pseudomallei were up-regulated in response to the presence of 0.05 μM L-Met. Notably, D-Met enhanced the susceptibility of B. pseudomallei H777 biofilm cells to ceftazidime. Our findings indicate a correlation between methionine supplementation and the up-regulation of nuclease genes, leading to eDNA depletion and the dispersal of preformed B. pseudomallei H777 biofilm. This enhances the susceptibility of biofilm cells to ceftazidime, showing promise in combating biofilm-associated B. pseudomallei infections.

TRNA anticodon cleavage by target-activated CRISPR-Cas13a effector.

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.

Structure of Escherichia coli exonuclease VII

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped β-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events.

Injectable Nano-Micro Composites with Anti-bacterial and Osteogenic Capabilities for Minimally Invasive Treatment of Osteomyelitis.

The effective management of osteomyelitis remains extremely challenging due to the difficulty associated with treating bone defects, the high probability of recurrence, the requirement of secondary surgery or multiple surgeries, and the difficulty in eradicating infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Hence, smart biodegradable biomaterials that provide effective and precise local anti-infection effects and can promote the repair of bone defects are actively being developed. Here, a novel nano-micro composite is fabricated by combining calcium phosphate (CaP) nanosheets with drug-loaded GelMA microspheres via microfluidic technology. The microspheres are covalently linked with vancomycin (Van) through an oligonucleotide (oligo) linker using an EDC/NHS carboxyl activator. Accordingly, a smart nano-micro composite called "CaP@MS-Oligo-Van" is synthesized. The porous CaP@MS-Oligo-Van composites can target and capture bacteria. They can also release Van in response to the presence of bacterial micrococcal nuclease and Ca2+, exerting additional antibacterial effects and inhibiting the inflammatory response. Finally, the released CaP nanosheets can promote bone tissue repair. Overall, the findings show that a rapid, targeted drug release system based on CaP@MS-Oligo-Van can effectively target bone tissue infections. Hence, this agent holds potential in the clinical treatment of osteomyelitis caused by MRSA.

Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast.

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo.

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) and are also thought to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and little is known about their functional activities and target specificities in vivo. Here, we describe five pAgos from mesophilic bacteria that act as programmable DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmids and chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum, can induce DNA interference between homologous sequences resulting in targeted processing of multicopy plasmid and genomic elements. EmaAgo also protects bacteria from bacteriophage infection, by loading phage-derived guide DNAs and decreasing phage DNA content and phage titers. Thus, the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

DECIPHERING THE ROLE OF GENE EDITING: RETORT FOR PERPLEXED UNHEALTHY AND DISEASED CONDITIONS

A breakthrough approach that unleashed the function of bacterial nucleases and the engineered ones has prompted the Creation of innovative gene editing technologies and procedures. In practically all eukaryotic cells, puzzled unclear Situations can be resolved by directly addressing certain genomic sequences and modifying them for use in various tactics. This expanding field of research has made this possibility possible. The ability to detect unique phenomena linked to the Genetic and epigenetic variables driving the development of disease have been enhanced through genome editing. It has majorly promoted the way for creating more precise cellular and animal models for elucidating pathological pathways and has shown remarkable potential in varying fields such as biotechnology, crop engineering, and biomedical science research Is under way. The creation of sophisticated methods based on nucleases, such as those connected to the CRISPR system, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and CRISPR-Cas-Associated nucleases, has greatly enhanced their practical utility in designing most promising biotechniques. Surprisingly, The applications of genome editing are leading to a variety of therapeutic and therapy options. This review will focus on Some applications and research that have revealed their implementation and strategies. Keywords: Epigenetic, Biotechnology, CRISPR, ZFN, TALENs, Nucleases. Cite this article- Shweta Sharma, Parul Singh, AkshitaTiwari, Faizan ul Haque, Vijaylaxmi Tripathi, Nupur Raghav, Shweta Sharma, Priyambada Kumari, 2023, “Deciphering the Role of Gene Editing: Retort for Perplexed Unhealthy and Diseased Conditions” journal of Science Innovations and Nature of Earth, Vol. 3(1), page- 46-51 www.doi.org/10.59436/https://jsiane.com/archives3/12/73

Structure and activity of a bacterial defense-associated 3'-5' exonuclease.

The widespread CBASS (Cyclic oligonucleotide-Based Anti-phage Signaling System) immune systems in bacteria protect their hosts from bacteriophage infection by triggering programmed cell death. Cap18 is a predicted 3′-5′ exonuclease associated with hundreds of CBASS systems with diverse regulators and effectors. However, the structure, biochemical activities, and biological roles of Cap18 remain unknown. Here we determine a high-resolution X-ray crystal structure of E. coli upec-117 Cap18, and show that it adopts a homodimeric structure similar to the bacterial DEDDh-family nucleases RNase T and Orn. We show that the protein is an active exonuclease with a preference for ssDNA over ssRNA, presenting non-sequence specific activity on both single-stranded and 3’ overhang DNA substrates. It does not require a cGAS-derived second messenger molecule for activity. Since E. coli upec-117 CBASS system can likely respond to stress signals other than phage infection through its WYL-domain transcription factor CapW, we are currently exploring the biological roles of Cap18 in modulating CapW responses using biochemical and cell-based assays. Together, these findings provide structural and functional insights into a previously uncharacterized enzyme associated with bacterial CBASS immune systems.

Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity

Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5’-end interactions. Unconventional interactions of PliAgo with the 5’-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity.

CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting dsDNA phage, ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities do not significantly impact infection outcomes.

Gene Editing: A Double-Edged Sword

This essay is about intrinsic planning parts that can alternate the enlarge of the particle that regulates our herbal cycles, the genome. Since the 1990s, first-class enchantment has been a focal factor of research. It commenced with the genome undertaking and will proceed to be an ambassador for the foreseeable future. The functions are many, and they are anticipated to have a significant speculative effect as properly as extraordinarily extreme hazards. The genome altering development trends have opened up the technique to truly zero in on and exchange genomic progressions in nearly all eukaryotic cells, whether or not they are planned or bacterial nucleases. Genome editing has loosened up our capacity to grant an explanation for the role of inherited qualities in infection with the aid of accelerating the development of increased right smartphone and models of animal of psychotic cycles, and it has begun to exhibit extraordinarily top achievable in a variety of fields, ranging from indispensable look up to utilized biotechnology and biomedical research. The late boom in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), file activator-like effector nucleases (TALENs), and assembled reliably interspaced quick palindromic repeat (CRISPR)– Cas-related nucleases, has accelerated the transition of fee from idea to medical practice. We observe the purposes of their subordinate reagents as quality-changing units in a range of human illnesses, and anticipated future medicines, which focuses on eukaryotic cells and animal models, in this evaluation of modern-day advances in the three critical genome-modifying propels (ZFNs, TALENs, and CRISPR/Cas9). Finally, we have a framework for clinical primers to use genome adjusting phases for sickness therapy, as nicely as some of the difficulties encountered throughout implementation.

Plasmon-assisted fast colorimetric detection of bacterial nucleases in food samples

We have developed a rapid, inexpensive, colorimetric and easy to use method able to detect living bacterial pathogens of zoonotic and foodborne interest. The method is based on detection of bacterial nucleases which cut selectively oligonucleotide probes that are designed in a way to induce aggregation of oligonucleotide stabilised gold-nanoparticles. We present the standardization of our method to detect nucleases secreted by Gram-positive (Staphylococcus aureus) and Gram-negative (Salmonella spp.) pathogens, not only in five different matrixes of food samples experimentally contaminated but also in naturally contaminated foodstuffs. Our method has shown sensitivity/specificity, detecting nucleases in less than two hours in supernatants from bacterial cell culture (1 CFU/mL) incubated for 15 h. The nucleases are detected by naked-eye inspection, and using minimal laboratory equipment. From a broader perspective, besides applications in foodstuff safety, we envision a potential use of our method to detect other bacterial and viral pathogens in the environment, and in veterinary and human health.

Efficiency of SpCas9 and AsCpf1 (Cas12a) programmable nucleases at genomic safe harbor loci in HEK293 cells

Rationale: The development of eukaryote genome engineering tools based on CRISPR-Cas programmable bacterial nucleases systems opens wide horizons for gene therapies, human disease cell modeling, as well as investigation into manifestation of disease phenotypes and visualization of cellular processes. The safety and approximation of experiments both at the cellular and organismal levels depend on the accuracy of introducing double-stranded breaks into the target DNA regions. The search for new variants of more accurate CRISPR-Cas nucleases and evaluation of their ability to hydrolyze nucleosome DNA in vivo is considered a critical task for the development of the genome engineering technologies.Aim: To analyze the activity of the programmable nuclease AsCpf1 (Cas12a), with low level of off-target activity, in the human genome loci that are safe for the introduction of transgenic constructs (“safe harbor”) and to compare its efficiency with that of the widely used SpCas9 nuclease in HEK293 cells.Materials and methods: We performed the bioinformatics analysis of the association between target regions with nucleosomes and other proteins in the safe harbor loci AAVS1 and GSH-Ch1 and the transcriptionally inactive gene MYBPC3 (cardiac myosin binding protein 3) based on ATAC-seq data for the HEK293FT cells obtained from the NCBI SRA database. Plasmids encoding SpCas9 and AsCpf1 nucleases and guide RNA to the target regions were constructed and transfected into the HEK293FT cells. Events in the target regions of the HEK293FT cell genome were studied in the sequenograms with the TIDE algorithm.Results: The results of the ATAC-seq experiments for HEK293FT cells have shown that the AAVS1 locus can be referred as open chromatin with a low nucleosome density, while the GSH-Ch1 locus can be attributed to closed chromatin. In HEK293FT cells, the cardiac MYBPC3 gene has intermediate chromatin density. Assessment of the efficiency of introducing breaks into the studied HEK293FT cell chromatin loci by nucleases has shown that SpCas9 is able to cope with chromatin of any nucleosome density, while AsCpf1 can effectively introduce DNA breaks only at loci with open chromatin, such as AAVS1 and MYBPC3. Editing events occur at a very low rate at the GSH-Ch1 locus with a high nucleosome density.Conclusion: We have found low efficiency of the AsCpf1 nuclease in the genomic safe harbor locus GSH-Ch1, which is characterized by a high nucleosome density. When planning an experiment on AsCpf1 nuclease genome editing, the epigenetic chromatin landscape and the nucleosome density should be considered, as well as chromatin opening substances should be used.

Rational Design and Experimental Analysis of Short-Oligonucleotide Substrate Specificity for Targeting Bacterial Nucleases.

An undecamer oligonucleotide probe based on a pair of deoxythymidines flanked by several modified nucleotides is a specific and highly efficient biosensor for micrococcal nuclease (MNase), an endonuclease produced by Staphylococcus aureus. Herein, the interaction mode and cleavage process on such oligonucleotide probes are identified and described for the first time. Also, we designed truncated pentamer probes as the minimum-length substrates required for specific and efficient biosensing. By means of computational (virtual docking) and experimental (ultra-performance liquid chromatography–mass spectrometry and matrix-assisted laser desorption ionization time-of-flight) techniques, we perform a sequence/structure–activity relationship analysis, propose a catalytically active substrate–enzyme complex, and elucidate a novel two-step phosphodiester bond hydrolysis mechanism, identifying the cleavage sites and detecting and quantifying the resulting probe fragments. Our results unravel a picture of both the enzyme–biosensor complex and a two-step cleavage/biosensing mechanism, key to the rational oligonucleotide design process.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Toward precise CRISPR DNA fragment editing and predictable 3D genome engineering.

Ever since gene targeting or specific modification of genome sequences in mice was achieved in the early 1980s, the reverse genetic approach of precise editing of any genomic locus has greatly accelerated biomedical research and biotechnology development. In particular, the recent development of the CRISPR/Cas9 system has greatly expedited genetic dissection of 3D genomes. CRISPR gene-editing outcomes result from targeted genome cleavage by ectopic bacterial Cas9 nuclease followed by presumed random ligations via the host double-strand break repair machineries. Recent studies revealed, however, that the CRISPR genome-editing system is precise and predictable because of cohesive Cas9 cleavage of targeting DNA. Here, we synthesize the current understanding of CRISPR DNA fragment-editing mechanisms and recent progress in predictable outcomes from precise genetic engineering of 3D genomes. Specifically, we first briefly describe historical genetic studies leading to CRISPR and 3D genome engineering. We then summarize different types of chromosomal rearrangements by DNA fragment editing. Finally, we review significant progress from precise 1D gene editing toward predictable 3D genome engineering and synthetic biology. The exciting and rapid advances in this emerging field provide new opportunities and challenges to understand or digest 3D genomes.

DNA targeting and interference by a bacterial Argonaute nuclease.

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3-5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6-10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood–CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

The iron\u2013sulphur cluster in human DNA2 is required for all biochemical activities of DNA2

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. DNA2 contains an iron-sulphur (FeS) cluster, conserved in eukaryotes and in a related bacterial nuclease. FeS clusters in DNA maintenance proteins are required for structural integrity and/or act as redox-sensors. Here, we demonstrate that loss of the FeS cluster affects binding of human DNA2 to specific DNA substrates, likely through a conformational change that distorts the central DNA binding tunnel. Moreover, we show that the FeS cluster is required for DNA2’s nuclease, helicase and ATPase activities. Our data also establish that oxidation of DNA2 impairs DNA binding in vitro, an effect that is reversible upon reduction. Unexpectedly, though, this redox-regulation is independent of the presence of the FeS cluster. Together, our study establishes an important structural role for the FeS cluster in human DNA2 and discovers a redox-regulatory mechanism to control DNA binding.

Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential.

Bacterial non-specific nucleases are ubiquitously distributed and involved in numerous intra- and extracellular processes. Although all nucleases share the basic chemistry for the hydrolysis of phosphodiester bonds in nucleic acid molecules, the catalysis comprises diverse modes of action, which offers great potential for versatile biotechnological applications. A major criterium for their differentiation is substrate specificity. Specific endonucleases are widely used as restriction enzymes in molecular biology approaches, whereas the main applications of non-specific nucleases (NSNs) are the removal of nucleic acids from crude extracts in industrial downstream processing and the prevention of cell clumping in microfabricated channels. In nature, the predominant role of NSNs is the acquisition of nutrient sources such as nucleotides and phosphates. The number of extensively characterized NSNs and available structures is limited. Moreover, their applicability is mostly challenged by the presence of metal chelators that impede the hydrolysis of nucleic acids in a metal ion-dependent manner. However, a few metal ion-independent NSNs that tolerate the presence of metal chelators have been characterized in recent years with none being commercially available to date. The classification and biotechnological potential of bacterial NSNs with a special focus on metal ion-independent nucleases are presented and discussed.Key Points • Bacterial phospholipases (PLD-family) exhibit nucleolytic activity. • Bacterial nucleases of the PLD-family are metal ion-independent. • NSNs can be used in downstream processing approaches.