Purification and characterization of the single‐strand‐specific and guanylic‐acid‐preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus

Abstract

An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The M r of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8.5 and 60 8C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn 21 , Ag 21 , Hg 21 , Fe 31 and Al 31 , inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA < 3 H AMP q RNA . dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5 H dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5 H dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5 H dCMP as a hydrolytic product, coupled with the resistance of (dC) 10 and deoxyribodinucleoside monophosphates having cytosine either at the 3 H or the 5 H end, indicates that C-linkages are resistant to cleavage by nuclease Bh1.Keywords: base specificity; Basidiobolus haptosporus; endoexonuclease; extracellular nuclease, single-strandspecific nuclease.Single-strand-specific nucleases are widely distributed in microorganisms, plants and animals. They exhibit high selectivity for single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids and hence are widely used as probes for the structural determination of nucleic acids, mapping mutations and studying the interactions of DNA with various intercalating agents [1]. They have also been implicated in recombination, repair [2] and replication [3]. Though singlestrand-specific nucleases are ubiquitously distributed, the enzymes of analytical interest originate mainly from fungal sources. However, only a few enzymes, such as S1 nuclease from Aspergillus oryzae, P1 nuclease from Penicillium citrinum, Bal 31 nuclease from Alteromonas espejiana, Neurospora crassa, Ustilago maydis and mung bean nucleases, have been sufficiently characterized [4]. Most of the analytically important single-strand-specific nucleases have an acid optimum pH and show an obligate requirement for metal ions to be active, which prevents their use in chelating buffers and in the presence of metal chelators. There is thus a need to look for single-strand-specific nucleases with opt...

Quantification, base composition, and fate of extracellular DNA in marine sediments

“…1996). Moreover, recent studies reported that extracellular nuclease produced by the fungus Basidiobolus haptosporus hydrolyzed DNA with preferential liberation of 5Ј-deoxyguanylate monophosphate, whereas DNA containing cytosine linkages were refractory to nuclease cleavage (Desai and Shankar 2000). In light of these findings, our results suggest that extracellular DNA in marine sediments might be selectively degraded, utilized, or both.…”

Purification and Characterization of Extracellular Nuclease from Bacillus Firmus VKPACU-1

“…Similar observations have been made in the case of Uracil specific ribonuclease from Bizionia sp which strongly supported the present observation [34]. Further Desai and Shankar [14] also have reported the highest activity of Bh1 nucleases from Basidiobolus haptosporus. The high sensitivity of the dsDNase activity towards low salt concentrations can be correlated to the suppression of localized melting by electrostatic stabilization of the DNA, especially the stabilization of AT rich regions in dsDNA [37].…”

“…Thus the present nuclease differs from other nucleases in pH stability by showing 100% single strand hydrolyzing activity between 6.5 and 8.0 and the dsDNA hydrolyzing activity between pH 6.0 and 8.0. Similar observations were also made for an extracellular nuclease from Basidiobolus haptosporus (Bh1), which showed high stability and retained a significant amount (65%) of its activity between pH 6.0 and 9.0 for 12 h at 37°C [14]. Salt concentration is known to affect the activity of single strand specific nucleases.…”

“…But a single strand specific nuclease from Rhizopus stolonifer showed no effect with thiol reagents at 5 mM concentaration [32]. In contrast, the activity of nuclease Bh1 was severely inhibited by Dithiothreitol and β-mercaptoethanol at 1 mM concentration [14] and GBSV1-NSN nuclease was slightly reduced [36]. It was found that the nuclease activity in this study was not inhibited by thiol reagents DTT or β-mercaptoethanol, suggesting that thiol groups are not essential for the activity of the enzyme.…”

Molecular and Functional Analyses of a Novel Class I Secretory Nuclease from the Human Pathogen, Leishmania donovani

“…DTT) abolished its nuclease activity. Furthermore, it has also been reported that some microbial members of the P1/S1 family are typically soluble, secreted enzymes (47)(48)(49)(50)(51), which function extracellularly to cleave RNA and single-stranded DNA without any apparent nucleo-base preference/specificity (reviewed in Ref. 58).…”