Efficient Purification of Nuclease P1 from Penicillium citrinum Using Polyethylene Glycol/Disodium Guanosine Monophosphate Aqueous Two-Phase System.

Nuclease P1 (NP1) can hydrolyze nucleic acids into four 5'-mononucleotides, which are widely used in the pharmaceutical and food industries. In this paper, an aqueous two-phase system (ATPS) was developed to purify NP1 from Penicillium citrinum. Polyethylene glycol (PEG) and nucleotides salts were studied to form ATPSs, among which PEG3000/disodium guanosine monophosphate (GMPNa2) was researched, including the phase composition and pH. Using 14% (w/w) PEG3000 and 20% (w/w) GMPNa2 ATPS at pH 5.0, the best recovery and purification factor, 82.4% and 3.59, were obtained. The recovery of NP1 was 98.3% by the separation of ultrafiltration from the PEG-rich phase. The recycling use of GMPNa2 was also studied, and 95.1% of GMPNa2 in the salt-rich phase was obtained with the addition of ethanol as the solvent. These results showed that the ATPS was effective for purification of NP1.

Stabilization of telomeric G-quadruplex by ligand binding increases susceptibility to S1 nuclease.

The extent of thermodynamic stabilization of telomeric G-quadruplex (G4) by isomers of G4 ligand L2H2-6OTD, a telomestatin analog, is inversely correlated with susceptibility to S1 nuclease. L2H2-6OTD facilitated the S1 nuclease activities through the base flipping in G4, unlike the conventional role of G4 ligands which inhibit the protein binding to DNA/RNA upon ligand interactions.

Mapping RNA with Nuclease S1.

This protocol provides details for nuclease S1 mapping of mRNA using a uniformly labeled, single-stranded DNA probe. DNA-RNA hybrids are generated, which are subsequently digested with nuclease S1. The digestion products are separated using gel electrophoresis and analyzed by radiography.

Analysis of RNA by Primer Extension.

For mapping the 5' termini of mRNA molecules, primer extension is the method of choice. A purified oligonucleotide is end-labeled using polynucleotide kinase. The probe and a population of mRNA are allowed to hybridize, and the primers and template are used to carry out reverse transcription using an enzyme cloned from the Moloney murine leukemia virus. The primer extension products are separated on a denaturing polyacrylamide gel and analyzed by radiography.

Mapping RNA.

Details of the fine structure of a particular transcript may be assessed by S1 mapping or ribonuclease protection. The use of either of these techniques allows the detection of the 5' and 3' ends of a particular mRNA, as well as the splice junctions, precursors, and processing intermediates of mRNA. Primer extension provides a measure of the amount of a particular mRNA species and allows an exact determination of the 5' end of the mRNA. These three methods are introduced here.

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

A label-free protamine-assisted colorimetric sensor for highly sensitive detection of S1 nuclease activity.

A label-free, sensitive, simple and general colorimetric method was reported to monitor S1 nuclease activity based on protamine-assisted aggregation of gold nanoparticles (AuNPs). Here, protamine, a linear polycation, was used as a medium for causing the aggregation of negatively charged AuNPs by electrostatic interactions, resulting in changes in the surface plasmon resonance (SPR) absorption bands as well as the color of AuNPs. Here, the AuNPs were employed as an indicator to detect the level of S1 nuclease in the solution. Substrate DNA could be cleaved into small fragments by the specific S1 nuclease, which effectively prevents the electrostatic interaction between DNA and protamine and thus facilitates the interaction between protamine and AuNPs. The quantitative analysis of S1 nuclease activity can be performed via directly measuring the changes in the absorption spectra of the AuNPs. Using S1 nuclease as a model analyte, the limit of detection was estimated to be 1.0 × 10-4 U mL-1. Furthermore, the proposed concept has been successfully applied in S1 nuclease analysis of serum samples, offering an ultrasensitive strategy for the speedy detection of the nuclease activity and providing a new avenue for high-throughput screening of nucleases and drugs with potential inhibition properties.

32P-Postlabeling Analysis of DNA Adducts.

32P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA, and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of 32P-orthophosphate from [γ-32P]ATP; chromatographic separation of labeled adducts, and detection and quantification by means of their radioactive decay. Using 10 µg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 109-1010 normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.

A genome-wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells.

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age-dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high-throughput replica-pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age-specific mutation suppression gene. While wild-type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells.

An alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease.

Recent studies show that NucS endonucleases participate in mismatch repair in several archaea and bacteria. However, the function of archaeal NucS endonucleases has not been completely clarified. Here, we describe a NucS endonuclease from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans (Tga NucS) that can cleave uracil (U)- and hypoxanthine (I)-containing dsDNA at 80 °C. Biochemical evidence shows that the cleavage sites of the enzyme are at the second phosphodiester on the 5'- site of U or I, and at the third phosphodiester on the 5'-site of the opposite base of U or I, creating a double strand break with a 4-nt 5'-overhang.The ends of the cleaved product of Tga NucS are ligatable, possessing 5'-phosphate and 3'-hydroxyl termini, which can be utilized by DNA repair proteins or enzymes. Tga NucS displays a preference for U/G- and I/T-containing dsDNA over other pairs with U or I, suggesting that the enzyme is responsible for repair of U and I in DNA that arise from deamination. Biochemical characterization of cleaving U- and I-containing DNA by Tga NucS was also investigated. The DNA-binding results show that the enzyme exhibits a higher affinity for normal, U- and I-containing dsDNA than for normal, U- and I-containing ssDNA. Therefore, we present an alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease in hyperthermophilic archaea.

Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts.

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2'-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.

Endonucleases responsible for DNA repair of helix-distorting DNA lesions in the thermophilic crenarchaeon Sulfolobus acidocaldarius in vivo.

The DNA repair mechanisms of hyperthermophiles can provide important insights for understanding how genetic information is maintained under extreme environments. Recent biochemical studies have identified a novel endonuclease in hyperthermophilic archaea, NucS/EndoMS, that acts on branched DNA substrates and mismatched bases. NucS/EndoMS is thought to participate in the DNA repair of helix-distorting DNA lesions, including UV-induced DNA damage and DNA adducts, and mismatched bases; however, the specific in vivo role of NucS/EndoMS in hyperthermophilic archaeal DNA repair has not been reported. To explore the role of this protein, we knocked out the nucS/endoMS gene of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. While the nucS/endoMS-deleted strain exhibited sensitivity to DNA adducts, it did not have high mutation rates or any sensitivity to UV irradiation. It has been proposed that the XPF endonuclease is involved in homologous recombination-mediated stalled-fork DNA repair. The xpf-deficient strain exhibited sensitivity to helix-distorting DNA lesions, but the sensitivity of the nucS/endoMS and xpf double knockout strain did not increase compared to that of the single knockout strains. We conclude that the endonuclease NucS/EndoMS works with XPF in homologous recombination-mediated stalled-fork DNA repair for the removal of helix-distorting DNA lesions in S. acidocaldarius.

Electrochemical aptasensor for sulfadimethoxine detection based on the triggered cleavage activity of nuclease P1 by aptamer-target complex.

Herein, a simple and selective electrochemical method was developed for sulfadimethoxine detection based on the triggered cleavage activity of nuclease P1 by the formation of aptamer and sulfadimethoxine conjugate. After probe DNA was immobilized on gold electrode surface, aptamer DNA labeled with biotin at its 5'-terminal was then captured on electrode surface through the hybridization reaction between probe DNA and aptamer DNA. The formed double-stranded DNA (dsDNA) can block the digestion activity of Nuclease P1 towards the single-stranded probe DNA. Then, the anti-dsDNA antibody was further modified on electrode surface based on the specific interaction between dsDNA and antibody. Due to the electrostatic repulsion effect and steric-hindrance effect, a weak electrochemical signal was obtained at this electrode. However, in the presence of sulfadimethoxine, it can interact with aptamer DNA, and then the formation of dsDNA can be blocked. As a result, the probe DNA at its single-strand state can be digested by Nuclease P1, which leads to the failure of the immobilization of anti-dsDNA antibody. At this state, a strong electrochemical signal was obtained. Based on the change of the electrochemical signal, sulfadimethoxine can be detected with linear range of 0.1-500 nmol/L. The detection limit was 0.038 nmol/L. The developed method possesses high detection selectivity and sensitivity. The applicability of this method was also proved by detecting sulfadimethoxine in veterinary drug and milk with satisfactory results.

Artificial Restriction DNA Cutter Using Nuclease S1 for Site-Selective Scission of Genomic DNA.

By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool. © 2019 by John Wiley & Sons, Inc.

Heterologous expression and characterization of Penicillium citrinum nuclease P1 in Aspergillus niger and its application in the production of nucleotides.

Nuclease P1 gene (nuc P1) which was cloned from Penicillium citrinum and expressed in A. niger Bdel4 with the low-background extracellular protein. The expression strategy of multi-copy nuc P1 in the A. niger with the linker of 2A peptide was applied to improve the enzyme activity of nuclease P1, the highest activity up to 77.6 U/mL. After Ni-chelate purification, the specific enzyme activity, the optimum temperature and pH were 32.4 U/mg, 65 °C and 5.3 respectively. The recombination nuclease P1 was activated by addition of Mg2+, Zn2+ and Cu2+, and inhibited by addition of Ca2+, Fe2+, Mn2+, Ni2+, Co2+, Mg2+, K+ and EDTA. Furthermore, the enzyme hydrolyses yeast RNA efficiently into 5'- nucleotides. Through enzymolysis, the highest concentration of nucleotides achieved 15.12 mg/mL, and 75U nuclease P1 is suitable amount should be added to the enzymolysis system.

Distinct roles of XPF-ERCC1 and Rad1-Rad10-Saw1 in replication-coupled and uncoupled inter-strand crosslink repair.

Yeast Rad1-Rad10 (XPF-ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1-Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1-Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1-Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1-Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1-Rad10 removes ICL via NER, whereas in S/G2, Rad1-Rad10 facilitates NER-independent replication-coupled ICLR.

Enzyme-Regulated DNA-Based Logic Device.

Herein, we report a carbazole (Cz) ligand that displays distinct turn-on fluorescence signals upon interaction with human telomeric G-quadruplex ( h-TELO) and nuclease enzymes. Interestingly, Cz selectively binds and stabilizes the mixed hybrid topology of h-TELO G-quadruplex that withstands digestion by exonucleases and nuclease S1. The distinct fluorescence signatures of Cz-stabilized h-TELO with nucleases are used to design conceptually novel DNA devices for selectively detecting the enzymatic activity of DNase I as well as performing logic operations. An INHIBIT logic gate is constructed using h-TELO and DNase I as the inputs while the inputs of h-TELO and nuclease S1 form a YES logic gate. Furthermore, a two-input two-output reusable logic device with "multireset" function is developed by using h-TELO and DNase I as inputs. On the basis of this platform, combinatorial logic systems (INHIBIT-INHIBIT and NOR-OR) have been successfully installed using different combinations of nucleases as inputs. Moreover, this new strategy of using a synthetic dual emissive probe and enzyme/DNA inputs for constructing reusable logic device may find important applications in biological computing and information processing.

Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

Characteristics and application of S1-P1 nucleases in biotechnology and medicine.

3'-nucleases/nucleotidases of the S1-P1 family (EC 3.1.30.1) are single-strand-specific or non-specific zinc-dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single-strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure-function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.

The effects of DNA supercoiling on G-quadruplex formation.

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation.