Single-strand-specific nucleases are multifunctional enzymes and widespread in distribution. Their ability to act selectively on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids has led to their extensive application as probes for the structural determination of nucleic acids. Intracellularly, they have been implicated in recombination, repair and replication, whereas extracellular enzymes have a role in nutrition. Although more than 30 single-strand-specific nucleases from various sources have been isolated till now, only a few enzymes (S1 nuclease from Aspergillus oryzae, P1 nuclease from Penicillium citrinum and nucleases from Alteromonas espejiana, Neurospora crassa, Ustilago maydis and mung bean) have been characterized to a significant extent. Recently, some of these enzymes have been cloned, their crystal structures solved and their interactions with different substrates have been established. The detection, purification, characteristics, structure-function correlations, biological role and applications of single-strand-specific nucleases are reviewed.

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...