Endo-exonuclease of Aspergillus nidulans

Abstract

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.

Sugar non-specific endonucleases

“…PCC 7120 [33], Syncephalastrum racemosum [34], Cunninghamella echinulata [38], Streptomyces antibioticus [39] and Rhizopus stolonifer [40] are extracellular in nature. Endonucleases of mitochondrial origin have been reported from N. crassa [11,24], S. cerevisiae [28,29,31] and Aspergillus nidulans [32]. In the cases of N. crassa [24] and A. nidulans [32] their presence has been shown in vacuoles, conidia, mycelia and nuclei.…”

“…Endonucleases of mitochondrial origin have been reported from N. crassa [11,24], S. cerevisiae [28,29,31] and Aspergillus nidulans [32]. In the cases of N. crassa [24] and A. nidulans [32] their presence has been shown in vacuoles, conidia, mycelia and nuclei. Additionally, in N. crassa, the mitochondrial nuclease has been shown to occur as membrane-bound as well as soluble forms in approximately equal proportions [59].…”

“…Other a¤nity adsorbents include deoxythymidine 3P,5P-diphosphate (pdTp)-aminophenyl-Sepharose [79], NADP-agarose [80] and 5P-AMPagarose [81]. The a¤nity of sugar non-speci¢c endonucleases for single-stranded nucleic acids has been utilized for the puri¢cation of endonucleases of S. cerevisiae [30], A. nidulans [32], D. melanogaster [53] and Leishmania sp.…”

“…In A. nidulans, the 28-kDa endo^exonuclease is formed due to the proteolytic digestion of the 38-kDa polypeptide via the formation of a 33-kDa intermediate. Moreover, crude extracts, obtained from cells grown in the presence of PMSF, showed the presence of an immunoreactive 90-kDa polypeptide which is a putative precursor of the 28-kDa endo^exonuclease [32]. In N. crassa 90% of the intracellular neutral ssDNase activity is associated with endo^exonucleases localized in mitochondria, vacuoles and nuclei.…”

“…Many of the metal requiring endonucleases show synergism when metal ions are used in combination. For example, nucleases from S. aureus [105], A. espejiana [12], A. nidulans [32], shrimp hepatopancreas [52], mitogenic factor of S. pyogenes [36] and S. antibioticus (18-kDa nuclease) [39] exhibited maximum activity in the presence of Ca 2 and Mg 2 .…”