Endo-exonuclease of Aspergillus nidulans.
Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.
Koa
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Department of Biochemistry, McGill University, Montreal, Que., Canada.
Fraser
M J
MJ
Käfer
E
E
D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D004271:DNA, Fungal / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004720:Endonucleases; D005092:Exonucleases; C020104:endoexonuclease