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M J Fraser

Publications and source records attributed to M J Fraser.

At least 73 records · Page 4Linked to original sources
PubMed1980

Nucleases and their control in wild-type and nuh mutants of Neurospora.

A review of all of the work on Neurospora nucleases strongly suggests that five nucleases, originally isolated on the basis of markedly different properties, may actually be derived from a single inactive precursor polypeptide via different routes of proteolysis. One of these nucleases may be involved in DNA repair and/or recombination. Two repair-deficient mutants of Neurospora, uvs-3 and nuh-4, may have a lesion in protease(s) which control the level of this nuclease or in some function which regulates the protease(s). Both of these mutants map in the same gene region and they may be defective in recombination, since they are sensitive to various mutagens and to mitomycin C and they show high frequency of spontaneous, but not radiation-induced, recessive lethal mutations and/or deletions.

DNA Repair↗
PubMed1979

Alkaline deoxyribonucleases released from Neurospora crassa mycelia: two activities not released by mutants with multiple sensitivities to mutagens.

Three major alkaline deoxyribonuclease (DNase) activities have been identified in sorbose-containing liquid culture medium in which wild-type Neurosporacrassa 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 RNA; DNase B, a Mg++-dependent single-strand 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.

Calcium↗
PubMed1979

The major intracellular alkaline deoxyribonuclease activities expressed in wild-type and Rec-like mutants of Neurospora crassa.

Over 95% of the deoxyribonuclease (DNase) activity of log-phase mycelia of Neurospora crassa is expressed as single-strand (ss) specific endonucleolytic activity. This activity is associated with three nucleases (D1, D2, and D3) which after partial purification from extracts, express activity with double-strand (ds) DNA as well. All three enzymes also degrade RNA at approximately the same rates that they degrade ss-DNA. D3 has been identified as endoexonuclease, an enzyme previously shown to have endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA, both of which are inhibited by ATP. D3 is inhibited by ATP, is relatively resistant to p-hydroxymercuribenzoate (PHMB), and sediments with an apparent molecular weight of 75 000. D2 has the properties of the previously described mitochondrial nuclease. It is a relatively unstable Mg2+-dependent endonuclease with no appreciable strand specificity for DNA. In addition, it is not inhibited by ATP and is strongly inhibited by PHMB and by the ethylenediamine tetraacetic acid (EDTA). It also sediments with an apparent molecular weight of 75,000. The properties of D1 are quite variable from one preparation to another. Freshly isolated D1 sediments with an apparent molecular weight of 180 000. It often shows some inhibition by ATP, but is relatively resistant to both PHMB and EDTA. However, on 'ageing,' the properties of D1 gradually convert to those of D2 with concomitant decrease in molecular weight, loss of inhibition by ATP, and increase in sensitivities to PHMB and EDTA. The results indicate that D1 is very likely a second form of the mitochondrial enzyme. Evidence was obtained for the presence of protein inhibitor(s) in crude extracts which may account for the masking of the ds-DNase activities of these enzymes in extracts. Two Rec-like mutants of Neurospora (uvs-3, and nuh-4) are deficient mainly inexpressed levels of D3, the endo-exonuclease. However, the levels of inactive endo-exonuclease precursor in these two mutants are higher than in the wild type. There may, therefore, be some defect in the conversion of precursor to active enzyme in these two mutants. Another mutant, which is not sensitive to mutagens relative to the wild (nuh-3), has depressed levels of both endo-exonuclease and the mitochondrial enzyme. Nuh-3 has some defect in the conversion of D1 to D2. Proteinases probably play some role in vivo in these enzyme conversions.

Deoxyribonucleases↗
PubMed1978

Neurospora endoexonuclease and its inactive (precursor?) form.

Two nuclease activities which were shown previously to copurify from extracts of log-phase Neurospora mycleia, a single-strand specific endonuclease activity (with DNA and RNA), and a strand nonspecific exonuclease activity (with DNA only) have been found to be associated with a single polypeptide. The enzyme has therefore been classified as an endoexonuclease. In logphase extracts, about 75% of this enzyme was found to exist in an inactive form which was activated in vitro either by endogenous phenylmethylsulfonyl fluoride sensitive proteinase(s) or by exogenous trypsin. The inactive form of endoexonuclease has been purified 45-fold in 15% yield free of the active enzyme. On electrophoresis in 6 M urea--polyacrylamide gels, it migrated at a much slower rate than the active enzyme, indicating that it is a less acidic and(or) larger protein than the active nuclease. The strong adsorption of this inactive enzyme on octyl-Sepharose suggests that the protein may have a relatively large hydrophobic domain. The protein may be a precursor of the active enzyme (a pronuclease) or a strong complex of enzyme with a proteinaceous inhibitor that is not dissociated in 6 M urea or during a variety of chromatographic procedures.

DNA, Single-Stranded↗
PubMed1976

A second form of the single-strand specific endonuclease of Neurospora crassa which is associated with a double-strand exonuclease.

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.

Adenosine Triphosphate↗
PubMed1975

Enzymes of Neurospora crassa which attack UV-irradiated DNA.

Two excision-deficient mutants of Neurospora crassa contain normal levels of two enzymes, a single-strand-specific exonuclease and a single-strand specific endonuclease, which attack UV-irradiated DNA. Mutants of N. crassa have been obtained in which the activity of the latter enzyme as well as an activity with native DNA are simultaneously affected. These activities are also apparently low in excision-proficient uvs3 strain of N. crassa which has many of the characteristics of the recA mutants of E. coli.

DNA↗
PubMed1974

Isolation of repeated and self-complementary sequences from E. coli DNA.

We have used the single-strand specific nuclease from Neurospora crassa and chromatography on methylated albumin-kieselguhr to purify and characterize repeated and self-complementary sequences from Escherichia coli DNA. Approximately 0.5% of the genome renatures spontaneously at zero time and another 2% renatures somewhat more rapidly than the total DNA. The early renaturing DNA has a base composition and a Tm similar to the total DNA and contains on the average 100 base pairs; the self-complementary DNA also has a base composition like E. coli but contains a mean of 170 base pairs. No evidence was obtained for the presence of a highly redundant sequence.

Base Composition↗