Properties Of Nuclease Research Articles

A novel naturally occurring tripyrrole with potential nuclease and anti-tumour properties

The DNA targeting and membrane damaging activities of a novel tripyrrole 1 obtained as a red pigment from the Micrococcus sp. were investigated. It was found that compound 1 binds with DNA efficiently and facilitates copper-mediated DNA cleavage as well as peroxidation of membrane lipids by a process that does not require any external reducing agent. Compound 1 also showed impressive cytotoxicity to both mouse and human tumour cell lines. The membrane damaging ability of compound 1 might be vital in its nuclease and cytotoxicity properties. Interestingly, compared to the various DNA cleaving agents, compound 1 showed a preferential binding with the G–C rich domain.

Designer Zinc Finger Proteins: Tools for Creating Artificial DNA-Binding Functional Proteins

The design of artificial functional DNA-binding proteins has long been a goal for several research laboratories. The zinc finger proteins, which typically contain many fingers linked in tandem fashion, are some of the most studied DNA-binding proteins. The zinc finger protein's tandem arrangement and its the ability to recognize a wide variety of DNA sequences make it an attractive framework to design novel DNA-binding peptides/proteins. Our laboratory has utilized several design strategies to create novel zinc finger peptides by re-engineering the C(2)H(2)-type zinc finger motif of transcription factor Sp1. Some of the engineered zinc fingers have shown nuclease and catalytic functional properties. Based on these results, we present the design strategies for the creation of novel zinc fingers.

Pressure perturbation calorimetic studies of the solvation properties and the thermal unfolding of staphylococcal nuclease

A rather new technique, pressure perturbation calorimetry (PPC), was applied to study volumetric and solvation properties of staphylococcal nuclease (Snase) in its native and unfolded state with high precision. Furthermore, the effects of various chaotropic and cosmotropic co-solvents on the solvation and unfolding behaviour of Snase was investigated in detail. In PPC, the apparent coefficient of thermal expansion of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent at the protein–solvent interface. In the native state, the protein shows a very strong thermal expansion of 1.0×10−3 K−1 at 10°C, which decreases steeply to 0.65×10−3 K−1 at 40°C. This behaviour is discussed in terms of a continuous release of condensed water from the protein surface. Upon unfolding, the volume decreases by about 19 mL×mol−1. Solutions of the cosmotropic and chaotropic compounds glycerol, sorbitol, K2SO4 and urea, respectively, show characteristic deviations from the thermal expansion and volumetric properties of the pure buffer solution. The solvent contribution to the apparent coefficient of thermal expansion of the protein, α, is enhanced considerably when the protein is immersed in a solvent known to be more structured than H2O (even the more structured D2O has a drastic effect) and nearly eliminated in a solvent in which “normal” water is largely absent (e.g., in 1.5 M urea). Similarly to D2O, a continuous increase in solvation was observed with increase in glycerol or sorbitol content in the buffer, which leads to an increase in protein stability, as is verified by the increasing Tm and ΔH values obtained by microcalorimetric measurements (DSC). In this regard, sorbitol is the more efficient agent. The reduction of ΔV in the presence of these stabilisers can in part be attributed to the formation of a partial unfolded state of the protein, in part it is due to the temperature dependence of ΔV. The preferential binding of urea reduces the hydration level, also in the native state, causing the protein to approach a more disordered state at high urea concentration. The increase in ΔV and the decrease in ΔH with increasing urea concentration support these conclusions. A stabilising effect, even though there is a reduction in solvation around the protein is observed for 0.5 M K2SO4 as co-solvent. In this case, surprisingly, ΔV is found to be positive which is an indication of the formation of a swollen, molten globule kind of unfolded state at the transition. Finally, ΔV values for the temperature-induced unfolding are compared with corresponding data for the pressure-induced unfolding of Snase.

Properties of and Substrate Determinants for the Exonuclease Activity of Human Apurinic Endonuclease Ape1

Ape1 is the major human abasic endonuclease, initiating repair of this common DNA lesion by incising the phosphodiester backbone 5′ to the damage site. This enzyme also functions in specific contexts to excise 3′-blocking termini, e.g. phosphate and phosphoglycolate residues, from DNA. Recently, the comparatively “minor” 3′ to 5′ exonuclease activity of Ape1 was found to contribute to the excision of certain 3′-mismatched nucleotides. In this study, I characterize more thoroughly the 3′-nuclease properties of Ape1 and define the effects of specific DNA determinants on this function. Data within shows that Ape1 is a non- or poorly processive exonuclease, which degrades one nucleotide gap, 3′-recessed, and nicked DNAs, but exhibits no detectable activity on blunt end or single-stranded DNA. A 5′-phosphate, compared to a 5′-hydroxyl group, reduced Ape1 degradation activity roughly tenfold, suggesting that the biological impact of certain DNA single strand breaks may be influenced by the terminal chemistry. In the context of a base excision repair-like DNA intermediate, a 5′-abasic residue exerted an about tenfold attenuation on the 3′ to 5′ exonuclease efficiency of Ape1. A 3′-phosphate group had little impact on Ape1 exonuclease activity, and oligonucleotides harboring these blocking termini were activated by Ape1 for DNA polymerase β extension. Ape1 was also found to remove 3′-tyrosyl residues from 3′-recessed and nicked DNAs, suggesting a potential role in processing covalent topoisomerase I–DNA intermediates formed during chromosome relaxation. While exhibiting preferential excision of thymine in a T:G mismatch context, Ape1 was unable to degrade a triple 3′-thymine mispair. However, Ape1 was able to excise double nucleotide mispairs, apparently through a novel 3′-flap-type endonuclease activity, again activating these substrates for polymerase β extension.

Synthesis, structure and nuclease properties of several ternary copper(II) peptide complexes with 1,10-phenanthroline

Three new ternary peptide–Cu(II)–1,10-phenanthroline (phen) complexes, [Cu( l-ala–gly)(phen)]·3.5H 2O 1, [Cu( l-val–gly)(phen)] 2 and [Cu(gly– l-trp)(phen)]·2H 2O 3, have been prepared and structurally characterised. These compounds exist as distorted square pyramidal complexes with the five co-ordination sites occupied by the tridentate peptide dianion and the two heterocyclic nitrogens of the phenanthroline ligand. The bulk of the lateral chain in the peptide moiety determines the relative disposition of the phen ligand. Thus, in [Cu( l-val–gly)(phen)] 2, the phenanthroline plane is deviated towards the opposite side of the isopropyl group of the l-valine moiety. On the other hand, in [Cu(gly– l-trp)(phen)]·2H 2O 3 the absence of stacking interactions between phen and indole rings and the presence of an intramolecular CH⋯π interaction should be pointed out. These complexes exhibit significant differences in their nuclease activity which depends on the nature of the peptidic moiety, the complex [Cu(gly– l-trp) (phen)]·2H 2O 3 being the most active.

Degradation of Single Stranded Nucleic Acids by the Chemical Nuclease Activity of the Metal Complex [Cu(phen)(nal)]+

The chemical design of metal complexes of the type [Cu(phen)(antib)]+ (where antib is a quinolone or a fluoroquinolone) has been carried out in an approach to better understand how the coordination of their components affect the activity of quinolones. The ability of [Cu(phen)(nal)]+ to interact with DNA in vivo and its capacity to promote the degradation of plasmid and chromosomal DNA, under reductive conditions has been previously reported. However whether this compound utilizes other intracellular targets to promote bacterial killing was a question that deserved to be answered. In this paper, the studies of the chemical nuclease properties encoded by the metal complex [Cu(phen)(nal)]+ were extended by using different types of single chain nucleic acids, i.e, ribosomal and tumor mosaic virus RNAs as well as poly-dA-dT. Our results showed that degradation of the nucleic acids occurred only under reductive conditions. Although MPA and [3-mercaptoethanol were the chemical reducers that best assisted the nuclease reaction, other biological compounds such as citric and succinic acid also were shown to act like reducers in that reaction. All.hough the nuclease activity of [Cu(phen)(nal)]+ was comparable to that exhibited by bis copper phenanthroline [Cu(phen)z]2+our results showed that none of the individual components of [Cu(phen)(nal)]+ was able to promote the degradation of either the RNAs or poly(dA-dT). These results strongly support the hypothesis that the metal complex [Cu(phen)(nal)] uses not only DNA but also RNA as targets to promote bacterial killing.

Influence of the a-ring on the redox and nuclease properties of the prodigiosins: importance of the bipyrrole moiety in oxidative DNA cleavage.

Prodigiosin (Prod, 1) is the parent member of a class of polypyrrole natural products that exhibit promising immunosuppressive and cytotoxic activity. They can facilitate copper-promoted oxidative double-strand (ds) DNA cleavage through reductive activation of Cu(II). This is triggered by oxidation of the electron-rich Prod molecule and may provide a basis for the cytotoxicity of the prodigiosins. To gain an understanding of this activity, we prepared several Prod analogues with various A-ring systems to examine their electrochemical properties in acetonitrile (MeCN) as a means to establish a basis for structure-reactivity relationships in copper-promoted nuclease activity. The intact bipyrrole (BP) chromophore is critical for the copper-mediated nuclease properties of the Prods. In fact, simple BP systems are shown to facilitate oxidative single-strand (ss) DNA cleavage. Replacement of the Prod A-pyrrole ring with alternative arenes (phenyl, furan-2-yl, or thiophen-2-yl) inhibits DNA strand scission and raises the half-peak oxidation potential (E(p/2)) of the Prod free base [E(p/2) = 0.44 V vs saturated calomel electrode (SCE) in MeCN] by ca. 200 mV. The same effect was achieved through attachment of an electron-withdrawing group (acetyl) at the 5'-position of the A-pyrrole ring. The structural modifications that inhibit DNA cleavage correlate with known structure-reactivity relationships of Prods against leukemia and melanoma cancer cells. The implications of our findings with regard to the cytotoxicity of the Prods are discussed.

Molecular interactions of human Exo1 with DNA.

Human Exo1 is a member of the RAD2 nuclease family with roles in replication, repair and recombination. Despite sharing significant amino acid sequence homology, the RAD2 proteins exhibit disparate nuclease properties and biological functions. In order to identify elements that dictate substrate selectivity within the RAD2 family, we sought to identify residues key to Exo1 nuclease activity and to characterize the molecular details of the human Exo1-DNA interaction. Site-specific mutagenesis studies demonstrate that amino acids D78, D173 and D225 are critical for Exo1 nuclease function. In addition, we show that the chemical nature of the 5'-terminus has a major impact on Exo1 nuclease efficiency, with a 5'-phosphate group stimulating degradation 10-fold and a 5'-biotin inhibiting degradation 10-fold (relative to a 5'-hydroxyl moiety). An abasic lesion located within a substrate DNA strand impedes Exo1 nucleolytic degradation, and a 5'-terminal abasic residue reduces nuclease efficiency 2-fold. Hydroxyl radical footprinting indicates that Exo1 binds predominantly along the minor groove of flap DNA, downstream of the junction. As will be discussed, our results favor the notion that the single-stranded DNA structure is pinched by the helical arch of the protein and not threaded through this key recognition loop. Furthermore, our studies indicate that significant, presumably biologically relevant, differences exist between the active site dynamics of Exo1 and Fen1.

Plasmid RK2 ParB protein: purification and nuclease properties.

The parCBA operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown. In this study ParB was overexpressed by cotranslation with ParC in Escherichia coli by using a plasmid construct that contained the parC and parB genes under the control of the T7 promoter. Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography. Sizing-column analysis indicated that ParB exists as a monomer in solution. Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures. ParB also exhibits 5'-->3' exonuclease activity. This ParB activity on a 5'-end-labeled, double-stranded DNA substrate produces a 3', 5'-phosphorylated dinucleotide which is further cleaved to a 3', 5'-phosphorylated mononucleotide. The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined.

The measurement of exonuclease activities by atomic force microscopy

We have applied atomic force microscopy (AFM) to the measurement of BAL 31 nuclease activities. BAL 31 nuclease, a species of exonuclease, is used to remove unwanted sequences from the termini of DNA before cloning. For cutting out only the appropriate sequences, it is important to know the nuclease properties, such as digestion speed and the distribution of the lengths of the digested DNA. AFM was used to obtain accurate measurements on the lengths of DNA fragments before and after BAL 31 nuclease digestion. We analyzed 4 DNAs with known number of base pairs (288, 778, 1818, and 3162 base pairs) for correlating the contour length measured by AFM with the number of base pairs under the deposition conditions used. We used this calibration for analyzing DNA degradation by BAL 31 nuclease from the AFM measurement of contour lengths of digested DNAs. In addition, the distribution of digested DNA could be analyzed in more detail by AFM than by electrophoresis, because digested DNA were measured as a population by electrophoresis, but were measured individually by AFM. These results show that AFM will be a useful new technique for measuring nuclease activities.

Generation of 5,5-Dimethyl-1-pyrrolineN-Oxide Hydroxyl and Scavenger Radical Adducts from Copper/H2O2Mixtures: Effects of Metal Ion Chelation and the Search for High-Valent Metal–Oxygen Intermediates

A metal-catalyzed nucleophilic addition mechanism for the formation of radical adducts of the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) has been described recently (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990,Biochem. Biophys. Res. Commun.172, 1073–1080; P. M. Hanna, W. Chamulitrat, and R. P. Mason, 1992,Arch. Biochem. Biophys.296, 640–644). In the present investigation, we have demonstrated that the recently reported inhibition of copper-dependent hydroxyl radical formation by the complexing agent 1,10-phenanthroline (OP), which appears to contradict the well-known chemical nuclease properties of CuI(OP)2, is an artifact resulting from an inhibition of the nucleophilic addition of water to DMPO by OP (A. C. Mello-Filho and R. Meneghini, 1991,Mutat. Res.251, 109–113). Copper bound to OP was found to be a good catalyst of hydroxyl radical formation: the CuII(OP)2complex can be reduced by H2O2and the CuI(OP)2generated reacts with the peroxide to form[formula]In contrast, no evidence could be obtained for oxidant formation from the[formula]reaction system, despite the detection of a prominent signal from the DMPO hydroxyl radical adduct (DMPO/[formula](the formation of which was due solely to the nucleophilic addition of water to DMPO). The failure to generate an oxidant in this reaction mixture was attributed to the failure of hydrogen peroxide to reduce[formula]as hydroxyl radical formation did occur when[formula]was added directly to H2O2. However, in order to account for the high concentration of α-hydroxyethanol radi cals detected when ethanol was included in the[formula]reaction, the possibility that an oxidant in addition to[formula](e.g., CuO+) is generated is discussed.

Fluorescence dynamics of staphylococcal nuclease in aqueous solution and reversed micelles

The dynamical fluorescence properties of the sole tryptophan residue (Trp-140) in Staphylococcus aureus nuclease (EC 3.1.31.1) have been investigated in aqueous solution and reversed micelles composed of either sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane or cetyltrimethylammonium chloride (CTAC) in isooctane/hexanol (12:1 by volume). The fluorescence decay of nuclease in the different environments can be described by a trimodal distribution of fluorescence lifetimes at approx. 0.5, 1.5 and 5.0 ns. The relative amplitudes depend on the environment. For pH 9.0 solutions the contribution of the two shortest lifetime components in the distribution is largest for AOT and smallest for CTAC reversed micelles. There is reasonable agreement between the average fluorescence lifetime and the fluorescence quantum efficiency confirming a significant fluorescence quenching in AOT reversed micelles. Fluorescence anisotropy decay revealed that the tryptophan environment in aqueous nuclease solutions is rigid on a nanosecond timescale. When nuclease was entrapped into reversed micelles the tryptophan gained some internal flexibility as judged from the distinct presence of a shorter correlation time. The longer correlation time reflected the rotational properties of the protein-micellar system. Modulation of the overall charge of nuclease (isoelectric point pH 9.6) by using buffer of pH 9.0 and pH 10.4, respectively, and of the size of empty micelles by selecting two values of the water to surfactant molar ratio, had only a minor effect on the rotational properties of nuclease in the positively charged reversed micelles. Encapsulation of nuclease in anionic reversed micelles resulted in the development of protein bound to aggregated structures which are immobilised on a nanosecond timescale. According to far UV vircular dichroism results the secondary structure of nuclease only followed the already published pH-dependent changes. Encapsulation had no major effect on the overall secondary structure.

Nuclease properties of two putative zinc finger peptides

We studied the interaction of wheat germ 5S rRNA with synthetic polypeptides whose amino acid sequences were similar to that of the second zinc finger of Xenopus laevis transcriptional factor IIIA (TFIIIA). The results clearly show that in addition to weak 5S rRNA binding activity (data not shown), these two 30 amino acid long polypeptides hydrolyse some phosphodiester bonds of wheat germ 5S rRNA. The cleavage pattern of plant 5S rRNA is very specific and the cuts occur only after the pyrimidine residues. The same properties of these peptides were furthermore observed for E. coli tRNA Phe. We found that the digestion specificity of both the zinc finger peptides is very similar to that of a pancreatic ribonuclease (RNase A).

Effects of amino acid substitutions on the pressure denaturation of staphylococcal nuclease as monitored by fluorescence and nuclear magnetic resonance spectroscopy.

In the present study we have used high hydrostatic pressure coupled with either time-resolved and steady-state fluorescence or NMR spectroscopy in order to investigate the effects of amino acid substitutions on the high-pressure denaturation properties of staphylococcal nuclease. This protein has been shown previously to be structurally heterogeneous in its native state. On the NMR time scale, four distinct interconverting conformational forms arise from the population of both cis and trans Xaa-Pro peptide bonds (His46-Pro47 and Lys116-Pro117) [Evans et al. (1989) Biochemistry 28, 362; Loh et al. (1991) in Techniques in Protein Chemistry II, pp 275-282, Academic Press, New York]. Mutations in the protein sequence have been shown to change the distribution among the various forms [Alexandrescu et al. (1989) Biochemistry 28, 204; Alexandrescu et al. (1990) Biochemistry 29, 4516]. Time-resolved fluorescence on a series of mutants with altered equilibria for cis/trans isomerism about the 116-117 peptide bond did not reveal any simple relationship between the position of the cis/trans equilibrium in the folded state and the heterogeneity of the fluorescence decay. However, the specific dynamic properties of each mutant, as revealed by time-resolved fluorescence, do appear to be correlated with their partial molar volume changes of denaturation. A striking finding is that mutation of either (or both) of the prolines that exhibits structural heterogeneity to glycine greatly alters the stability of the protein to pressure. These mutations also result in decreased chain mobility as assessed by time-resolved fluorescence. It appears that packing defects, which allow for peptide bond cis/trans heterogeneity in the wild-type protein, are removed by the Pro-->Gly substitutions.

Influence of lectin concentration on the catalytic properties of S1 nuclease bound to concanavalin A-sepharose

Partially purified S1 nuclease was bound through its carbohydrate moiety to Con A-Sepharose containing increasing amounts of lectin. The retention of activity was high, varying essentially from 75% on the "low lectin" matrix (1 mg Con A/mL of Sepharose), to no detectable activity on the "high lectin" matrix (8 mg Con A/mL of Sepharose). However, approximately 50% activity could be restored in "high lectin" matrix when the coupling was carried out in the presence of glucose, suggesting that the loss of activity on the "high lectin" matrix is caused by conformational changes brought about by the multiple attachment of the enzyme to the matrix. Interaction of Con A with S1 nuclease was used to predict the nature of carbohydrate moiety and its location with respect to the active site of the enzyme. Immobilization resulted in an increase in the optimum temperature, pH, and temperature stabilities, but it did not affect the pH optimum. A marginal increase in the apparent Km was observed. The bound enzyme also showed enhanced stability toward 8 M urea. On repeated use, the bound enzyme retained more than 80% of its initial activity after 6 cycles. These results are discussed taking into consideration the factors affecting immobilized enzymes. In addition, the potential use of immobilized S1 nuclease as an analytical tool is discussed.

Biochemical properties and hormonal regulation of barley nuclease.

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.

The Release and Some Properties of Nuclease from Various Pollen Species

Summary Mature pollen grains of all 15 species examined release nuclease within the first minutes of contact with a liquid medium. These species include plants with both binucleate and trinucleate pollen and with biotic and abiotic modes of pollination. The enzyme of Pinus nigra pollen exhibits similar characteristics as the earlier described enzyme of tobacco pollen classified as plant nuclease I (E.C. 3.1.30.x). It is shown to be a sugar-unspecific endonuclease with maximal activity at acidic pH and preference for single-stranded molecules. The enzyme produces oligonucleotides and 5'-mononucleotides. Nuclease of the other pollen species also has more or less pronounced preference for denatured as opposed to native DNA. A pH in the range of 5.0-5.9 and 6.8-7.0 is optimal for the enzyme of dicotyledons and cereal species, respectively. Different pollen species release different amounts of nuclease with different specific activities. The enzyme also exhibits species-related variation in electrophoretic mobility and in the number of molecular forms. Molecular sieving of pine pollen diffusate produced one nuclease peak with an apparent molecular mass of 29.5 x 10 3 .

Purification and properties of extracellular nuclease from tobacco pollen

Proteins diffusing from tobacco pollen grains exhibit different phosphohydrolytic activities. Molecular sieving produces nuclease fractionation into forms I, II and III with apparent molecular masses ≥ 60 × 103, 32.9 × 103 and 24.6 × 103, respectively, and separation of principal forms II and III from phosphatase and major part of 5′- and 3′-nucleotidase activities. These forms did not differ in the mode of substrate attack and were combined for further enzyme characterization.

Differentiation of staphylococci on the basis of nuclease properties.

The quantity, thermostability, and serological pattern of nucleases produced by different staphylococci were studied. Staphylococcal strains were isolated from nine different species of animals or from humans. Staphylococcus aureus, Staphylococcus intermedius, and Staphylococcus hyicus subsp. hyicus were vigorous producers of nuclease, whereas the coagulase-negative staphylococci, except S. hyicus subsp. hyicus, produced significantly less nuclease. The nucleases of all strains were found to be thermostable. S. aureus, S. intermedius, and S. hyicus subsp. hyicus could be distinguished from each other and from coagulase-negative staphylococci on the basis of inhibition of nuclease activity by specific antibodies.

32] Purification and properties of S1 nuclease from Aspergillus

Publisher Summary This chapter explains the purification and properties of S1 nuclease from Aspergillus. The most convenient way to distinguish double helical from single-stranded nucleic acids is by digestion of the single-stranded components with specific nucleases. Several ribonucleases, notably pancreatic RNase and RNase T1, have been routinely used to hydrolyze single-stranded RNA. Nucleases capable of specifically degrading single-stranded DNA have been characterized only more recently. Among this class of nucleases is the single-strand specific nuclease from Aspergillus oryzae, which has come to be known as S1. The advantages of S1 are that it can be prepared easily and cheaply in large quantities, that it is stable to low concentrations of denaturants often used in annealing and that its specificity for single-stranded nucleic acids is extremely high under the fight conditions. The chapter presents a highly reproducible partial purification of S1 yielding enzyme that is perfectly adequate for all uses involving precipitating and quantitating nucleic acids from a mixture of double and single-stranded species treated with S1.