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
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.