BAL 31 nuclease as a probe in concentrated salt for the B-Z DNA junction

The BAL 31 nuclease, an extracellular nuclease from A. espejiana, specifically recognizes and cleaves the salt induced conformational junction between B and Z-DNA. Short segments of (dC-dG) left-handed Z-helix, comprising approximately 1% of the total DNA, are specifically detected within two different recombinant plasmids. The BAL 31 enzyme is highly resistant to inactivation by the presence of high concentrations of a variety of electrolytes that stabilize left-handed helices, is active at physiological pH, and can be used to probe both linear and circular DNAs. Additionally, the nuclease cleaves left-handed (dC-dG)n only very poorly, if at all. Thus, the BAL 31 nuclease can be utilized as a probe for helical junctions and consequently for segments of left-handed DNA that might exist within predominantly right-handed naturally occurring genomes.     

Mechanism of action of Escherichia coli exonuclease III

Exonuclease III is the major apurinic/apyrimidinic (AP) endonuclease of Escherichia coli, accounting for more than 80% of the total cellular AP endonuclease activity. We have shown earlier that the endonucleolytic activity of exonuclease III is able to hydrolyze the phosphodiester bond 5' to the urea N-glycoside in a duplex DNA [Kow, Y. W., & Wallace, S. S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8354-8358]. Therefore, we were interested in studying the mechanism of action of the endonucleolytic activity of exonuclease III by preparing DNA containing different base lesions as well as chemically modified AP sites. When AP sites were converted to O-alkylhydroxylamine residues, exonuclease III was able to hydrolyze the phosphodiester bond 5' to O-alkylhydroxylamine residues. The apparent Km for different O-alkylhydroxylamine residues was not affected by the particular O-alkylhydroxylamine residue substituted; however, the apparent Vmax decreased as the size of the residue increased. On the basis of a study of the substrate specificity of exonuclease III, a modification of the Weiss model for the mechanism of action of exonuclease III is presented. Furthermore, a temperature study of exonucleolytic activity of exonuclease III in the presence of Mg2+ showed discontinuity in the Arrhenius plot. However, no discontinuity was observed when the reaction was performed in the presence of Ca2+. Similarly, no discontinuity was observed for the endonucleolytic activity of exonuclease III, in the presence of either Ca2+ or Mg2+. These data suggest that, in the presence of Mg2+, exonuclease III, in the presence of either Ca2+ or Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Precursor-product relationship of larger to smaller molecular forms of the BAL 31 nuclease from Alteromonas espejiana: preferential removal of duplex exonuclease relative to endonuclease activity by proteolysis

Two molecularly and kinetically distinct major species of the extracellular nuclease BAL 31 from Alteromonas espejiana, previously characterized as the "fast" (F) and "slow" (S) BAL 31 nucleases, have been evidenced to derive from proteolysis starting from a still larger (approximately 120 kDa) precursor nuclease. The expected protease activity in the culture fluid has been confirmed and is strongly dependent on the cell growth phase. The disappearance of the largest nuclease species with the concomitant sequential appearance of first the F and then the S species has been demonstrated for nuclease obtained from culture supernatants as a function of cell growth phase. Nuclease from periplasmic extracts displayed very little of the F and S nucleases. Treatment of purified F nuclease with Pronase or subtilisin readily converted it to species with only a few percent of the native exonuclease activity against duplex DNA but retaining much of the initial activity against single-stranded DNA. Electrophoresis in nuclease-detecting gels demonstrated a parallel conversion of the larger species to one indistinguishable in molecular weight from the S species. The observed loss of exonuclease activity could correspond to the conversion of the F to the S nuclease. However, treatment of S nuclease with subtilisin resulted in a drastic reduction of exonuclease activity of this enzyme on duplex DNA with retention of most of the activity against single-stranded and nicked circular duplex DNA substrates. Evidence of internal proteolysis of the S nuclease could be seen after electrophoresis in denaturing gels but only after the denaturation buffer was adjusted to 6 M in urea. The preferential removal of the exonuclease activity may enhance the usefulness of the BAL 31 nuclease in such applications as heteroduplex mapping.     

Sequence specificity of BAL 31 nuclease for ssDNA revealed by synthetic oligomer substrates containing homopolymeric guanine tracts

Background

The extracellular nuclease from Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 5′-mononucleotides, cleaves negatively supercoiled DNA to the linear duplex form, and cleaves duplex DNA in response to the presence of apurinic sites.

Principal Findings

In this work we demonstrate that BAL 31 activity is affected by the presence of guanine in single-stranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine''s presence in minimal homopolymeric tracts in the middle of short oligomer substrates and also by its presence at the 3′ end of ten and twenty base oligomers. G•C rich regions in dsDNA are known to cause a decrease in the enzyme''s nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access. Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G•C rich regions. Thus the presence of a guanine tract per se impairs the enzyme''s functionality, possibly due to the tract''s bulky nature and preventing efficient progression through the active site.

Conclusions

This study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA) oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.     

Isolation and comparison of two molecular species of the BAL 31 nuclease from Alteromonas espejiana with distinct kinetic properties

The extracellular nuclease from Alteromonas espejiana sp. BAL 31 can be isolated as two distinct proteins, the "fast" (F) and "slow" (S) species, both of which have been purified to homogeneity. The F and S species of the nuclease have molecular weights, respectively, of 109 X 10(3) and 85 X 10(3), and both are single polypeptide chains with an isoelectric pH near 4.2. Both species catalyze the degradation of single-stranded and linear duplex DNAs to 5'-mononucleotides. The degradation of linear duplex DNA occurs through a terminally directed hydrolysis mechanism that results in the removal of nucleotides from both the 3' and 5' ends. Apparent Michaelis constants (Km) have been obtained for the exonuclease activities of both species and for the activity against single-stranded DNA of the S species. The Km for the hydrolysis of single-stranded DNA catalyzed by the F species has not been obtained because the reaction velocity was maximal even at the lowest substrate concentrations accessible in the photometric assay. The ratio of the turnover numbers for the exonuclease activities of the two species indicates that the F species will shorten linear duplex DNA at a rate 27 +/- 5 (S.D.) times faster than an equimolar concentration of the S species in the limit of high substrate concentration, while the corresponding ratio for the activities against single-stranded DNA (1.2 +/- 0.1) shows that the two species are similar with respect to hydrolysis of this substrate. In the limit of high substrate concentrations, the F and S species break phosphodiester bonds in single-stranded DNA at rates 1.3 +/- 0.3 and 33 +/- 2 times those for the exonucleolytic degradation of linear duplex DNA, respectively. It has not been established whether the two species are physically related.     

Terminally directed hydrolysis of duplex ribonucleic acid catalyzed by a species of the BAL 31 nuclease from Alteromonas espejiana

The extracellular nuclease activities of Alteromonas espejiana sp. BAL 31 are mediated by at least two distinct protein species that differ in molecular weights and catalytic properties. The two species that have been purified to homogeneity and characterized, the "fast" (F) and "slow" (S) enzymes, both possess an exonuclease activity that shortens both strands of duplex DNA, with the F nuclease displaying a much greater (approximately 19-fold) turnover number for this degradation than the S species. In the present article, it is shown that the F species also mediates the terminally directed hydrolysis of a linear duplex RNA, gradually shortening molecules of this substrate through a mechanism that results in the removal of nucleotides from both the 3' and the 5' ends. This degradation proceeds with very infrequent introduction of scissions away from the termini as demonstrated by gel electrophoretic examination of the products of partial degradation, both in duplex form and after denaturation by reaction with CH3HgOH, and by electron microscopic characterization of duplex partially degraded molecules. The apparent Michaelis constant and turnover number have been determined. At equimolar enzyme concentrations in the limit of high substrate concentration, the F nuclease will degrade duplex RNA at a rate 0.021 +/- 0.010 (S.D.) times that for a duplex DNA of comparable guanine + cytosine content. The S species, by contrast, shows very little activity against the duplex RNA substrate relative to that of the F enzyme.     

Circular intermediates with missing nucleotides in the conversion of supercoiled or nicked circular to linear duplex DNA catalyzed by two species of BAL 31 nuclease

The extracellular nucleases from Alteromonas espejiana BAL 31 can catalyze the endonucleolytic and/or exonucleolytic hydrolysis of duplex DNA in response to a variety of alterations, either covalent or noncovalent, in DNA structure. The nuclease can exist as at least two kinetically and molecularly distinct protein species. The two species that have been studied, called the 'fast' (F) and 'slow' (S) nucleases, both readily convert negatively supercoiled DNAs to linear duplex molecules and accomplish this conversion through the formation of a circular duplex intermediate containing usually a single interruption in one strand. It is further shown that most of these intermediates contain gaps arising from the removal in a processive manner of one or more nucleotide residues after the introduction of the initial strand break (nick). Considering only the intermediates with gaps, the average number of missing residues is 6.3 +/- 0.5 and 2.8 +/- 0.3, respectively, for DNA acted upon by the F and S enzymes independently of the extent of conversion of supercoiled DNA. The nicks and gaps are bounded by 3'-hydroxyl and 5'-phosphoryl termini. When singly nicked circular DNA is used as the substrate, conversion to the linear duplex form occurs predominantly through a gapped circular intermediate with the same average numbers, within experimental error, of missing nucleotides for the respective nuclease species as found when supercoiled DNA is the substrate. The conversion to linear duplex DNA is much slower when nicked circular DNA is the substrate compared to that found when supercoiled DNA is the starting material.     

Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family

Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.     

Single-strand nuclease action on heat-denatured spermiogenic chromatin.

The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.     

A single apurinic site can elicit BAL 31 nuclease-catalyzed cleavage in duplex DNA

The extracellular nuclease from Alteromonas espejiana BAL 31 is a highly sensitive endonucleolytic probe for lesions that distort the helical structure of duplex DNA. The nuclease can be isolated as two distinct molecular species, the 'fast' (F) and 'slow' (S) species, which have different kinetic properties. When nonsupercoiled, covalently closed circular phage PM2 DNA containing apurinic sites introduced by heating at acid pH was incubated with individual fractions from a chromatographic column which separated the two nuclease species, cleavage of the DNA was observed which was greatly in excess of control levels using nonmodified DNA. The initial rates of such cleavage clearly paralleled the peaks of both absorbance and nuclease activity against single-stranded and linear duplex substrates. When samples of apurinic DNA were incubated with pooled fractions from the same column representing pure F and S nucleases, respectively, the rate and extent of the cleavage observed was dependent upon the average number of apurinic sites per molecule. Cleavage was readily detectable in samples containing an average of 1.1 apurinic sites per molecule with both species of the enzyme. These results indicate that either species of the BAL 31 nuclease can recognize and cleave in response to a single apurinic site in duplex DNA. The F nuclease appears to be approx. 2.5-times as efficient in cleaving DNA containing apurinic lesions as the S enzyme in extended incubations.     

Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion

A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of the pi-pi electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA.     

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.     

Heterogeneity of chromatin fragments produced by micrococcal nuclease action.

Digestion of calf thymus chromatin with micrococcal nuclease produces a mixture of apparently well defined nucleoprotein fragments which have been partially resolved by sedimentation on linear (5-20%) sucrose gradients. Sedimentation patterns reveal a predominant peak at the 11S position, three slower components, which have not previously been reported, at the 3.4S, 5.3S and 8.6S positions, and three faster components at the 17S, 22S and 26S positions. DNA isolated from the 3S to 12S region of gradients has been resolved on polyacrylamide gels into nine to ten discrete components ranging from 47 to 156 base pairs in length. A nearly identical pattern of small DNA products was obtained from chromatin digested in intact nuclei. These data suggest that chromatin contains either several types of subunits or predominently a single type of subunit which can be asymmetrically cleaved at any one of four or more sites.     

DNA-tropic inhibitors of nuclease action on DNA in liver nuclei

Distamycin is a potent, wide-spectrum inhibitor of the breaking of both free and intranuclear DNA with DNAse I and with own nuclear nucleases. It compares very favourably with actinomycin D, proflavin and ethidium bromide, especially in the inhibition of DNAse I action in the nuclei. This seems likely to be due to partial overlapping of the binding sites of the nuclei with chromatin proteins in contrast to distamycin that interacts with a minor furrow of DNA being blocked to a less extent by proteins. The DNA-tropic agents under test exert no qualitative effect on the kinetics of intranuclear DNA splitting by DNAse I. Carminomycin and bleomycin are the least effective inhibitors in all the systems depicted.     

Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system

Single-nucleotide polymorphisms (SNPs) in double-stranded DNA (dsDNA) have been straightforwardly genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide nucleic acid (PNA), a DNA analog, was used as a probe molecule. In its presence, genomic dsDNA was first treated with exonuclease III and then with nuclease S1. By these one-pot reactions, single-stranded DNA fragments including the SNP sites were formed in situ. These fragments were directly analyzed by MALDI-TOF MS, and the identity of the DNA base at the SNP site was determined in terms of mass number. By using two or more PNA probes simultaneously, multiplex analysis was also successful. Various genotypes of apolipoprotein E gene (ε2/ε2, ε3/ε3, ε4/ε4, ε2/ε3 and ε3/ε4) were identified from dsDNA obtained by PCR from corresponding patients.     

Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos

The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis. The chromatin containing these genes was examined by nuclease sensitivity. An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes. The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4. Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages.