Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea

Citation
V. Healy et al., Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea, BIOCHEM J, 339, 1999, pp. 713-720
Citations number
31
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL JOURNAL
ISSN journal
02646021 → ACNP
Volume
339
Year of publication
1999
Part
3
Pages
713 - 720
Database
ISI
SICI code
0264-6021(19990501)339:<713:PCACCO>2.0.ZU;2-M
Abstract
We have purified an endo-exonuclease from the fruiting body of the basidiom ycete fungus Armillaria mellea by using an ethanol fractionation step, foll owed by two rounds of column chromatography. The enzyme had an apparent mol ecular mass of 17500 Da and was shown to exist as a monomer by gel-filtrati on analysis. The nuclease was active on both double-stranded and single-str anded DNA but not on RNA. It was optimally active at pH 8.5 and also exhibi ted a significant degree of thermostability. Three bivalent metal ions, Mg2 +, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibite d by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3'-5' (but not a 5'-3') exonuclease activity. It generated 5'-phospho monoesters on its products that, after a prolonged incubation, were hydroly sed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-bas ed approach. Peptide mapping of the purified enzyme generated patterns cons istent with the amino acid sequence coded for by the cloned cDNA. A BLAST s earch of the SwissProt database revealed that A. mellea nuclease shared sig nificant amino acid similarity with two nucleases from Bacillus subtilis, s uggesting that the three might constitute a distinct class of nucleolytic e nzymes.