RNase and DNase activities in the alfalfa and lentil grown in iso-osmotic solutions of NaCl and mannitol

Nucleolytic activities from two plants of Leguminosae family were determined in order to consider if the nucleases of plants which belong to the same family or to the same species responded in similar ways to stress conditions during growth. Growth parameters of both plants were examined in parallel. In detail, seedlings from two plants, alfalfa (Medicago sativa L. cv. Luzerne Euver) and lentil (Lens culinaris cv. Thessalia), showed significant differences in response to iso-osmotic solutions of NaCl (100 mmol · L⁻¹ solution equivalent to conductivity 8.0 dS m⁻¹) and mannitol (190 mmol · kg⁻¹). Plant height and dry weight of mannitol/NaCl-treated seeds in both plants were lower in comparison to controls (water). Mannitol stress reduced height and dry weight in alfalfa seedlings more than did NaCl. By contrast, lentil seedling growth was inhibited more by NaCl stress than mannitol. In addition, DNase and RNase response to mannitol stress differed in each plant compared to the controls. Mannitol stress induced a sharp increase in DNase- and RNase-specific activity during the initial stages of alfalfa seedlings' growth, followed by a decrease during subsequent days; in lentil seedlings, these activities were inhibited throughout the entire growth period. NaCl stress inhibited the above activities in both plants. After native electrophoresis on gels polymerized in the presence of DNA/RNA, the overall band intensities confirmed the above quantitative results of alfalfa RNase and DNase activity. In addition, the active gel analysis revealed that the decrease of nucleolytic activities in mannitol-treated alfalfa seedlings was mainly due to the strong reduction of acid nucleases. This is the first report of different non-ionic osmotic response of type I plant nucleases during seedlings' growth. In vitro, the addition of up to 300 mmol/L mannitol did not affect acid and neutral nuclease activity in enzyme preparations extracted, purified, and separated from control and mannitol-treated alfalfa seedlings.Our results suggest that plant nucleases responded in a different way to osmotic stress and ionic stress conditions during seedlings' growth.     

Study of rat brain tissue nucleases]

Acid and alkaline nucleases of the brain tissues have been identified and partially purified. Alkaline DNA-ase hydrolyzing denatured DNA at pH 8,0; alkaline RNA-ase having optimal activity at pH 8,0 and nuclease intensively hydrolyzing both DNA and RNA at pH 5.0. The molecular weights of these enzymes have been determined.     

Identification of nucleases related to nutrient mobilization in senescing cotyledons from French bean

The knowledge about the physiological function of plant nucleases is scarce besides that they have been involved in nucleic acid degradation related with programmed cell death processes. Cotyledons provide a suitable system to investigate this process and the changes associated to nutrient mobilization. Nuclease activities have been determined in French bean seedlings. The total nuclease activity in French bean cotyledons is lower than in embryonic axes; however, several nucleases were detected by in-gel nuclease activity assays with extracts from cotyledons of French bean and ssDNA as substrate. The nuclease activities induced during cotyledon senescence showed higher activity at neutral than at acidic pH. Five different nuclease genes belonging to S1/P1 family have been identified in French bean genome database named PVN1 to PVN5. Their relative expression in cotyledons has been determined from the start of imbibition to senescence, and three genes from this family showed expression in cotyledons. PVN1 was expressed during early stages of seedlings development, whereas PVN4 and PVN5 were expressed during cotyledons senescence. The removal of epicotyl in French bean seedlings resulted in a decrease in the activity and in the expression of the genes associated with the cotyledons senescence process, i.e. PVN4 and PVN5. At the same time, the mobilization of reserves in those cotyledons was slowed down. In the same way, the deficit in phosphate and nitrate during seedlings development led to an acceleration of induction of these genes at the same time that reserves were utilized early on the time. Therefore, the induction of PVN4 and PVN5, the two S1 nuclease genes involved in the process of cotyledon senescence, is related to nutrient mobilization, supporting a possible role for nucleic acids in nutrient recycling during cotyledon senescence.     

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N⁶-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.     

Isolation and characterization of nucleases from a clinical isolate of Serratia marcescens kums 3958

Two new extracellular nucleases, nucleases SM1 and SM2, were purified from the culture fluid of S. marcescens kums 3958, a fresh clinical isolate. The purification was carried out by the following steps; ammonium sulfate precipitation, and DEAE-cellulose and Sephadex G-100 column chromatography. At the final step, nucleases SM1 and SM2 were purified about 3,700- and 1,000-fold, respectively. They were free from phosphomonoesterase and phosphodiesterase activities. The pIs were 8.1 and 7.5 for nucleases SM1 and SM2, respectively. The molecular weight was estimated to be 35,000 for both enzymes by SDS-polyacrylamide disc gel electrophoresis. The results of amino acid analyses showed that both the threonine and serine contents were higher in nuclease SM2 than in SM1. Furthermore, nuclease SM1 was more stable than nuclease SM2 at 4 degrees C. The other properties of the two enzymes were similar; pH optimum (8.0), Mg2+ or Mn2+ for activation, and inhibition by chemical reagents such as EDTA and pyrophosphate. No significant difference was found in base specificity between nucleases SM1 and SM2. Both enzymes specifically degraded double-stranded homopolymers, especially poly(I). poly(C), as well as yeast RNA and calf thymus DNA. They hardly degraded, however, single-stranded homopolymers such as poly(dA), poly(G), and poly(U).     

Activities of nucleases in senescing daylily petals

The activity of nucleases during organ death was investigated using daylily petals (Hemerocallis hybrid cv. Stella d’Oro), in which the processes associated with senescence are rapid and clearly ordered. The number of nuclei with fragmented DNA as well as activities of various nucleases increase before certain other events that are related to senescence. Furthermore, DNA breakage and activities of nucleases occur earlier when senescence is accelerated by abscisic acid and occur later when senescence is retarded by cycloheximide. These results suggest that the activities of nucleases contribute to the senescence of daylily petals. Therefore, studying the regulation of nuclease gene expression may be useful for understanding components of the signal transduction system that leads to the death of these organs.     

Two-dimensional zymogram analysis of nucleases in Bacillus subtilis

A two-dimensional zymogram procedure for the analysis of nucleases is described. Isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) were compared as first dimensions in combination with sodium dodecyl sulfate (SDS) electrophoresis as the second dimension in analyzing nucleases in lysates of Bacillus subtilis. All renaturable nucleases detected following SDS electrophoresis alone were resolved in NEPHGE-SDS electrophoresis gels whereas, in IEF gels, most either were at the basic end or were not present in the second-dimension gels. This method of analysis has revealed a complexity in nuclease species in B. subtilis not previously recognized. Eighty-three discreet nuclease activities have been detected in B. subtilis lysates. Using purified deoxyribonuclease I (bovine pancreas), as little as 10 pg of nuclease can be detected.     

An immunochemical study of Neurospora nucleases

Nucleases derived from Neurospora crassa mycelia with neutral single-strand (ss) endodeoxyribonuclease activity have been examined by immunochemical techniques and by sodium dodecyl sulfate - DNA gel electrophoresis. All of the intracellular nucleases, which have different divalent metal ion requirements, different strand specificities with single- and double-strand DNA, different modes of action on DNA and RNA, and other distinguishing characteristics, are immunochemically related to Neurospora endo-exonuclease. The evidence indicates that these enzymes are derived from one or more related large, inactive (precursor?) polypeptides that are first converted to 75- to 80-kdalton active polypeptide(s) which are very protease sensitive. Further limited proteolysis results in the production of the various active forms of nuclease studied here. Some proteolytic conversions may occur in a controlled manner in vivo in different cell compartments, but others are very likely artifacts resulting from uncontrolled proteolysis during extraction and isolation. The intracellular forms of Neurospora endo-exonuclease are immunologically cross-active with ss-DNA-binding nucleases isolated from Aspergillus nidulans and Saccharomyces cerevisiae. They are not immunochemically related to two extracellular Neurospora nucleases, the pancreatic DNase-I-like DNase A and a ss-specific exonuclease, and they are also not related to other fungal and plant nucleases with ss-specific endonuclease activity such as the S1 nuclease of Aspergillus oryzae, the P1 nuclease of Penicillium citrinum, and mung bean nuclease.     

Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings.

An endonuclease was isolated from 5 days old Agropyron elongatum 8x = Elytrigia turcica McGuire seedlings. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-cellulose and Heparin Sepharose column. The final preparation, named nuclease A, gave a single band after silver staining had followed SDS-electrophoresis that was identified with nuclease activities. The enzyme also showed a single band after activity staining on gel polymerized in the presence of heat denatured DNA (ssDNA)/RNA. The Mr of native enzyme was 36 and the enzyme's moiety consisted of one polypeptide chain. Nuclease A activity was stimulated in the presence of Zn(2+) and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA > or = RNA; hence it was classified as a plant nuclease type I (EC 3.1.30.2). Synthetic homopolyribonucleotides were hydrolyzed in the order polyU > polyI > or = polyA > polyG > polyC. Nuclease A nicked the supercoiled plasmid DNA while it was incapable of hydrolyzing dinucleoside monophosphates. With regard to nuclease A base linkage specificity towards a synthetic 5'-(32)P labeled deoxydecanucleotide [5'-(32)P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap downward arrow G bond and then to Gp downward arrow T, Cp downward arrow A and Gp downward arrow G bonds while it was incapable of hydrolyzing the Cp downward arrow C bond. The substrate's products of nuclease A were oligonucleotides with the monoesterified phosphate at the 3' position. Nuclease A may perform a crucial function in the metabolism of nucleic acids during seedling growth and could be used as a biochemical tool for analysis of nucleic acids structure.     

Purification, characterization, and role of nucleases and serine proteases in Streptomyces differentiation. Analogies with the biochemical processes described in late steps of eukaryotic apoptosis.

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.     

Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases

It was previously shown (Paddenberg et al (1996) Eur J Cell Biol 69, 105 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47 - 54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.     

Initiation of DNA damage responses through XPG‐related nucleases

Lesion‐specific enzymes repair different forms of DNA damage, yet all lesions elicit the same checkpoint response. The common intermediate required to mount a checkpoint response is thought to be single‐stranded DNA (ssDNA), coated by replication protein A (RPA) and containing a primer‐template junction. To identify factors important for initiating the checkpoint response, we screened for genes that, when overexpressed, could amplify a checkpoint signal to a weak allele of chk1 in fission yeast. We identified Ast1, a novel member of the XPG‐related family of endo/exonucleases. Ast1 promotes checkpoint activation caused by the absence of the other XPG‐related nucleases, Exo1 and Rad2, the homologue of Fen1. Each nuclease is recruited to DSBs, and promotes the formation of ssDNA for checkpoint activation and recombinational repair. For Rad2 and Exo1, this is independent of their S‐phase role in Okazaki fragment processing. This XPG‐related pathway is distinct from MRN‐dependent responses, and each enzyme is critical for damage resistance in MRN mutants. Thus, multiple nucleases collaborate to initiate DNA damage responses, highlighting the importance of these responses to cellular fitness.     

Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.     

Isolation and characterization of cell-associated nucleases related to streptococcal extracellular deoxyribonuclease D.

A group of at least four distinct nucleases designated DcI through DcIV were isolated from cellular extracts of group A streptococcal strain S43 and shown to be antigenically similar to streptococcal extracellular deoxyribonuclease (DNase) D. These cellular endonucleases degraded single- and double-stranded deoxyribonucleic acid (DNA) as well as ribonucleic acid (RNA) to acid-soluble oligonucleotides. The products of digestion of DNA bore 5'-terminal phosphates, and in partial digests pdX-pdG linkages were most susceptible and pdA-pdX linkages were most resistant to nuclease action. The enzymes had pH optima of 8.0 to 8.5, were inhibited by NaCl, were unaffected by sulfhydryl modifying reagents, and absolutely required a divalent cation. Nucleases DcIII and DcIV were apparently hydrophobic in nature since they required the presence of detergents for migration on nondenaturing polyacrylamide gels. All four nucleases were electrophoretically distinct on such gels, from each other, and from DNase D. Molecular weights of DcI and DcII were similar to that of DNase D, suggesting that the mobility differences of these enzymes at least are reflections of differing net charges. It is suggested that the cellular nucleases represent a group of processing intermediates in the maturation and excretion of DNase D.     

Structural and functional insight into sugar-nonspecific nucleases in host defense

In prokaryotes, sugar-nonspecific nucleases that cleave DNA and RNA in a sequence-independent manner take part in host defense, as well as site-specific restriction enzymes. Examples include the periplasmic nuclease Vvn and the secreted nuclease ColE7, which degrade foreign nucleic acid molecules in the host periplasm and in the cytoplasm of foreign cells, respectively. Recently determined crystal structures of Vvn and ColE7 in complex with double-stranded DNA provide structural insight into nonspecific DNA interactions and cleavage by sugar-nonspecific nucleases. Both nucleases bind DNA at the minor groove through a common 'betabetaalpha-metal' endonuclease motif and primarily contact the DNA phosphate backbone, probably to avoid sequence-dependent base recognition. In eukaryotes, several apoptotic endonucleases that are responsible for DNA degradation in programmed cell death also contain a betabetaalpha-metal fold at the active site, suggesting that they may recognize and cleave DNA in a comparable way.     

Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

Targeted genome editing in pluripotent stem cells using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous end-joining (NHEJ). Thus, ZFNs are powerful tools for targeted genome engineering in a variety of mammalian cell types, including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). As a paradigm for genome editing in pluripotent stem cells, we describe the use of ZFNs in murine ESCs for generating knockout alleles by NHEJ without selection or by HR employing different selection schemes.     

Local and systemic wound-induction of RNase and nuclease activities in Arabidopsis: RNS1 as a marker for a JA-independent systemic signaling pathway

Induction of defense-related genes is one way in which plants respond to mechanical injury. We investigated whether RNases are involved in the wound response in Arabidopsis thaliana. As in other plant systems, several activities are induced with various timings in damaged leaves, stems and seedlings in Arabidopsis, including at least three bifunctional nucleases, capable of degrading both RNA and DNA, as well as RNS1, a member of the ubiquitous RNase T(2) family of RNases. The strong induction of RNS1 is particularly interesting because it occurs both locally and systemically following wounding. The systemic induction of this RNase indicates that members of this family may be involved in defense mechanisms in addition to their previously hypothesized functions in nutrient recycling and remobilization. Additionally, the systemic induction appears to be controlled independently of jasmonic acid, and the local induction of RNS1 and the nuclease activities are independent of both JA and oligosaccharide elicitors. Consequently, a novel systemic pathway, likely involving a third signal, appears to exist in Arabidopsis.     

Affinities of various nucleases to DNA-Sepharose under non-digestive conditions: survey for productive affinity chromatography

1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.     

Structural specificities of five commonly used DNA nucleases

Five commonly used nucleases were surveyed for their ability to distinguish among several different DNA backbone configurations. The digestion data suggest that: (1) DNAase I binds across the minor groove; whereas (2) nuclease S1 and (3) micrococcal nuclease bind to an exposed single strand; (4) copper/phenanthroline seeks a base-pair step; and (5) DNAase II requires just a stacked single strand of limited exposure. Only micrococcal nuclease is demonstrably base-specific, with a strong preference for T, A over C, G in any structural context.