Three major alkaline deoxyribonuclease (DNase) activities have been identified in sorbose-containing liquid culture medium in which wild-type Neurospora crassa were grown: DNase A, a Ca++-dependent endonuclease of molecular weight 65,000 daltons which has no specificity for single-or double-stranded DNA (ss-DNA or ds-DNA) and no activity with RIWA; DNase B, a Mg++-dependent singlestrand specific exonuclease of molecular weight 78,000 daltons active with both ss-DNA and RNA; DNase C, a divalent metal ion-dependent endo-exonuclease of molecular weight 65,000 having single-strand specific endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA. Three mutants which were shown previously to have wide spectra of sensitivities to mutagens, and which exhibited reduced release of DNase activity on sorbose-containing agar test plates (the Nuh phenotype), were deficient relative to the wild-type in the release of these major alkaline DNases into the liquid culture medium. The uvs-3 mutant released only small amounts of DNase A and DNase C; nuh-4 did not release detectable DNase C and released only a very low level of DNase B; uvs-6 released only a low level of DNase A. A nuh mutant (nuh-3) which is not mutagen sensitive relative to the wild-type released low levels of DNase B.On the other hand, an ultraviolet light-sensitive mutant (nuc-2) which does not have the Nuh phenotype was normal in the release of these DNases.

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Nucleases derived from Neurospora crassa mycelia with neutral single-strand (ss) endodeoxyribonuclease activity have been examined by immunochemical techniques and by sodium dodecyl sulfate - DNA gel electrophoresis. All of the intracellular nucleases, which have different divalent metal ion requirements, different strand specificities with single- and double-strand DNA, different modes of action on DNA and RNA, and other distinguishing characteristics, are immunochemically related to Neurospora endo-exonuclease. The evidence indicates that these enzymes are derived from one or more related large, inactive (precursor?) polypeptides that are first converted to 75- to 80-kdalton active polypeptide(s) which are very protease sensitive. Further limited proteolysis results in the production of the various active forms of nuclease studied here. Some proteolytic conversions may occur in a controlled manner in vivo in different cell compartments, but others are very likely artifacts resulting from uncontrolled proteolysis during extraction and isolation. The intracellular forms of Neurospora endo-exonuclease are immunologically cross-active with ss-DNA-binding nucleases isolated from Aspergillus nidulans and Saccharomyces cerevisiae. They are not immunochemically related to two extracellular Neurospora nucleases, the pancreatic DNase-I-like DNase A and a ss-specific exonuclease, and they are also not related to other fungal and plant nucleases with ss-specific endonuclease activity such as the S1 nuclease of Aspergillus oryzae, the P1 nuclease of Penicillium citrinum, and mung bean nuclease.

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.

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A second form of single-strand specific endonuclease, which is stable to heating up to 74 degrees C and does not bind strongly to phosphocellulose, has been partially purified from extracts of mycelia of wild-type Neurospora crassa. The endonuclease is associated with an equally heat-stable exonuclease which degrades linear but not circular double-stranded DNA and does not attack double-stranded RNA. The exonuclease probably also degrades single-stranded DNA. Both endonuclease and exonuclease activities are inhibited by 0.1-0.5 mM ATP. The exonuclease is preferentially inhibited by a variety of agents and preferentially inactivated by trypsin. A DNA-unwinding activity has also been detected in the nuclease preparation. Protease(s) present in the nuclease preparation destroy the DNA-unwinding and exonuclease activities on incubation at 37 degrees C, but do not affect the endonuclease activity. However, the heat-stability and chromatographic properties of the endonuclease are affected by this treatment. The altered properties of the endonuclease are very similar to those of the single-strand specific endonuclease which has been previously described. The combined nuclease activities of the unaltered preparational make up a putative recombination nuclease of N. crassa.

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Immunochemical cross-reaction between the endo-exonuclease of Neurospora crassa, an enzyme previously implicated in recombination and recombinational DNA repair, and the recC-encoded polypeptide of Esherichia coli has been detected by immunoblotting extracts of strains of E. coli having a deletion that includes the recBCD genes but carrying multicopy plasmids bearing all three of the recBCD genes or only one or two of these genes. It was predicted that homology would also be found at the amino acid sequence level between the recC polypeptide and both nuclear and mitochondrial endo-exonucleases of Saccharomyces cerevisiae, which cross-react with antibodies raised to the N. crassa endo-exonuclease. Since the gene for the S. cerevisiae mitochondrial enzyme, NUCI, has been cloned and sequenced and the predicted amino acid sequence is known, this sequence was aligned with the predicted amino acid sequence of the recC polypeptide. Extensive homology was found by aligning 306 of the 329 amino acids of the yeast mitochondrial nuclease sequence with the carboxy-terminal one-quarter of the amino acid sequence of the recC polypeptide.Possible roles for endo-exonuclease of Neurospora crassa in recombination and recombinational repair of nuclear and mitochondrial DNAs have been proposed previously, on the basis of strong similarities in the enzymic activities of this enzyme and those of the recBCD-encoded nuclease (exonuclease V) of Escherichia coli (4) and deficiencies in active endo-exonuclease in nuclei and mitochondria of the mutagen-sensitive uvs-3 mutant of Neurospora sp., which may be abnormal in recombination (12,15). In addition, an immunochemically related nuclear endo-exonuclease of 72 kilodaltons (kDa) was shown to be very deficient in the recombination and recombinational double-strand DNA break repair-deficient rad52 mutant of Saccharomyces cerevisiae (5, 6). The nuclear endo-exonuclease is encoded by an essential gene which has been cloned and is currently being sequenced (T. Y.-K. Chow, unpublished observations). A second endo-exonuclease, of 38 kDa, which is also immunochemically related to the N. crassa endo-exonuclease is present in the S. cerevisiae mitochondria (7). It is encoded by the nonessential NUCI gene, which has recently been cloned and sequenced (19).In contrast to E. coli exonuclease V, the N. crassa endo-exonuclease is sugar nonspecific, acting on both DNA and RNA, and although it is not ATP dependent or ATP stimulated in its actions, it does possess a distributive single-stranded DNA-specific endonuclease activity and a highly processive exonuclease activity with linear doublestranded DNA, like exonuclease V (4). The latter activity in vitro can generate long single-stranded tails and long gaps in duplex DNA, which would be potentially recombinogenic substrates if also generated in vivo. Under slightly different conditions in vitro, N. crassa endo-exonuclease has been shown recently to make site-specific single-and doublestrand breaks in linear, but not in covalently closed circular, V (17)....