Specific inhibitors of Neurospora endo-exonuclease

1989 ◽  
Vol 67 (9) ◽  
pp. 632-641 ◽  
Author(s):  
Zafer Hatahet ◽  
Murray J. Fraser
Keyword(s):  
Neurospora Crassa ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Exonuclease Activity ◽  
Endonuclease Activity ◽  
Average Molecular Mass ◽  
Strand Breaks ◽  
Heat Stable ◽  
First Order Kinetics ◽  
Pbr322 Dna

A heat-stable, trypsin-sensitive cytosolic inhibitor of Neurospora crassa endo-exonuclease has been purified 300-fold in 90% yield from crude extracts of mycelia. On electrophoresis in sodium dodecyl sulfate – polyacrylamide gels, it showed a relatively broad band corresponding to polypeptides with an average molecular mass of about 24 kDa. The protein inhibited the single strand specific endonuclease activity of endo-exonuclease noncompetitively, but the inhibition was not complete (maxima of 70–95%) in the presence of excess inhibitor. The inhibition of the double strand exonuclease activity was competitive and complete when inhibitor was present in excess. In addition, the inhibitor completely blocked the formation of site-specific double strand breaks and nicking by endo-exonuclease in linearized pBR322 DNA. The ribonuclease activity of endo-exonuclease was also inhibited by this protein. The inhibition of Neurospora endo-exonuclease was very specific. Although inhibition was seen also for an immunochemically related nuclease derived from mycelia of Aspergillus nidulans, no inhibition was observed for two other nucleases of Neurospora, nor for a wide variety of commercially available nucleases. A bound form of inhibitor was also partially purified from the cytosol of Neurospora in association with inactive, but trypsin-activable endo-exonuclease. Activity gel analysis showed that this was an endo-exonuclease–inhibitor complex. On heating at 80 °C, inhibitor was released from the complex with the same first order kinetics as inactivation of the trypsin-activable endo-exonuclease and in approximately equivalent amounts. The bound inhibitor was also 24 kDa in size, but completely inhibited both the endo- and exonuclease activities of endo-exonuclease. It was found unexpectedly that heat-inactivated endo-exonuclease stimulated the exonuclease activity of the active enzyme without affecting the endonuclease activity. The stimulated exonuclease activity was also completely blocked by inhibitor.Key words: Neurospora crassa endo-exonuclease, inhibitor, constitutive, cytosolic.

scholarly journals Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Pauline Chanut ◽  
Sébastien Britton ◽  
Julia Coates ◽  
Stephen P. Jackson ◽  
Patrick Calsou
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Exonuclease Activity ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Additional Mechanism ◽  
Strand Breaks ◽  
Rad51 Focus ◽  
Dna Resection

Abstract Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.

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

1976 ◽  
Vol 54 (11) ◽  
pp. 971-980 ◽  
Author(s):  
M. J. Fraser ◽  
R. Tjeerde ◽  
K. Matsumoto
Keyword(s):  
Neurospora Crassa ◽  
Heat Stability ◽  
Single Strand ◽  
Dna Unwinding ◽  
Endonuclease Activity ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Heat Stable ◽  
Double Stranded Dna ◽  
Heating Up

A second form of single-strand specific endonuclease, which is stable to heating up to 74 °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 °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 preparation make up a putative recombination nuclease of N. crassa.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals Estimation of Rearrangement Break Rates Across the Genome

2018 ◽  
Author(s):  
Christopher Hann-Soden ◽  
Ian Holmes ◽  
John W. Taylor
Keyword(s):  
Neurospora Crassa ◽  
Interval Graph ◽  
Double Strand Breaks ◽  
Genomic Rearrangements ◽  
Strand Breaks ◽  
Minimum Cover ◽  
A Genome ◽  
History Of ◽  
Cover Problem ◽  
Special Case

Genomic rearrangements provide an important source of variation, but reconstructing the history of rearrangements often has many solutions. We answer the question of where rearrangements occur by solving the simpler problem of estimating the rate of double-strand breaks at every site in a genome. This problem is a special case of the minimum cover problem for an interval graph. We implement this method as a Python program, BRAG, and use it to estimate break rates in the genome of Neurospora crassa. We find that more frequent rearrangement in the subtelomeres facilitates the evolution of novel genes.

scholarly journals The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally.

1990 ◽  
Vol 10 (12) ◽  
pp. 6454-6459 ◽  
Author(s):  
T J Fahrner ◽  
S L Carroll ◽  
J Milbrandt
Keyword(s):  
Growth Factor ◽  
Molecular Mass ◽  
Pc12 Cells ◽  
Receptor Gene ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Carboxy Terminal

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.

Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor

1987 ◽  
Vol 7 (1) ◽  
pp. 403-409
Author(s):  
R J Bram ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Binding Sites ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Sodium Dodecyl ◽  
Heat Stable ◽  
Tata Element ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

A protein that binds specifically to Saccharomyces cerevisiae centromere DNA element I was purified on the basis of a nitrocellulose filter-binding assay. This protein, termed centromere-binding protein 1 (CP1), was heat stable and renaturable from sodium dodecyl sulfate (SDS), and assays of eluates from SDS gels indicated a molecular weight of 57,000 to 64,000. An activity with similar specificity and stability was detected in human lymphocyte extracts, and analysis in SDS gels revealed a molecular weight of 39,000 to 49,000. CP1-binding sites occurred not only at centromeres but also near many transcription units, for example, adjacent to binding sites for the GAL4-positive regulatory protein upstream of the GAL2 gene in S. cerevisiae and adjacent to the TATA element of the adenovirus major late promoter. A factor (termed USF) that binds to the latter site and stimulates transcription has been isolated from HeLa cells by others.

Partial characterization of an abnormal lactate dehydrogenase isoenzyme, LDH-1ex, in serum from a patient with hepatocellular carcinoma.

1989 ◽  
Vol 35 (5) ◽  
pp. 844-848
Author(s):  
D L Kalpaxis ◽  
E E Giannoulaki
Keyword(s):  
Hepatocellular Carcinoma ◽  
Lactate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Heat Stable ◽  
Single Polypeptide ◽  
Polypeptide Chains

Abstract Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1ex, that on electrophoresis on 10-g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypeptide chains (monomers), each having a molecular mass of about 32,000 Da. LDH-1ex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-1ex showed no activity when lactate was omitted from the substrate solution or replaced by ethanol.

Macrozin, a Novel Storage Globulin From Seeds of Macrozamia communis L. Johnson

1984 ◽  
Vol 11 (2) ◽  
pp. 69 ◽  
Author(s):  
RJ Blagrove ◽  
GG Lilley ◽  
TJV Higgins
Keyword(s):  
Molecular Weight ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Optical Rotatory Dispersion ◽  
Molecular Weights ◽  
Neutral Ph ◽  
Cellulose Acetate Membranes ◽  
Dodecyl Sulfate ◽  
Α Helix ◽  
Polypeptide Chains

The isolation, characterization and amino acid composition are reported for macrozin, the major storage globulin found in seeds of Macrozamia communis. Electrophoresis of macrozin on cellulose acetate membranes at neutral pH resulted in a single broad band indicating limited charge heterogeneity. Isoelectric focusing under dissociating and reducing conditions showed this globulin to be composed of a family of polypeptide chains with apparent isoelectric points in the range pH 6.0-7.5. Sedimentation equilibrium studies showed that the main component purified by gel filtration in aqueous buffers at neutral pH has a molecular weight of 260 000 and a sedimentation coefficient S020.w = 10.9 S. This component dissociates in 8 M urea to yield subunits of molecular weight 126 000. Each subunit is composed of disulfide-bonded polypeptide chains of approximate molecular weight 44 000. The apparent molecular weights for the macrozin subunit and its constituent polypeptides were 130 000 and 46 000 from dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dimeric nature of the main oligomer in aqueous solution was confirmed by crosslinking the subunits with dithiobis(succinimidylpropionate); the presence of three polypeptide chains per subunit is inferred from the molecular weights. Optical rotatory dispersion and circular dichroism measurements suggest that macrozin is devoid of α-helix in its native conformation, but contains some 25% α-helix after incubation with sodium dodecyl sulfate.

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa

1978 ◽  
Vol 56 (10) ◽  
pp. 927-933 ◽  
Author(s):  
W. S. Lin ◽  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamine Synthetase ◽  
Neurospora Crassa ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Affinity Column ◽  
Subunit Structure ◽  
Α Helix

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate–Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate α-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360 000 and as having a sedimentation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate α-helix content of 23–24%. The subunit generated by treatment with urea was found to be 45 000 daltons by gel filtration methods and a molecular weight of 46 000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45 000 daltons.

scholarly journals The opsonic fragment of the third component of human complement (C3).

1975 ◽  
Vol 141 (6) ◽  
pp. 1329-1347 ◽  
Author(s):  
T P Stossel ◽  
R J Field ◽  
J D Gitlin ◽  
C A Alper ◽  
F S Rosen
Keyword(s):  
Disulfide Bonds ◽  
Human Peripheral Blood ◽  
Sodium Dodecyl ◽  
Complement C3 ◽  
Coated Particles ◽  
Heat Stable ◽  
Fresh Serum ◽  
Dodecyl Sulfate ◽  
Autoradiographic Analysis ◽  
Oil Red O

Human peripheral blood phagocytes ingest Escherichia coli 026:B6 lipopolysaccharide (LPS)-coated paraffin oil droplets containing Oil red O only if fresh serum deposits C3 on the surfaces of the particles (opsonizes them), by reactions involving the properdin system. The rate of binding of purified [125-I]C3 in serum to LPS-coated particles correlated precisely with the rate of acquisition of ingestibility assayed spectrophotometrically. Once opsonized, LPS-coated particles remained fully ingestible and retained fixed [125-I]C3 radioactivity even after exposure to extremes of temperature, pH, ionic strength, phospholipases, urea or guanidine, some nonionic and ionic detergents, and organic solvents. Trypsin, human conglutinogen-activating factor, another heat-stable activity found in human serum, and sodium dodecyl sulfate removed radioactivity and diminished ingestibility of the opsonized particles. Alkylation, reduction plus alkylation and F(ab')2 from anti-C3 blocked ingestibility but did not alter particle-bound radioactivitymelectrophoretic and tryptic peptide autoradiographic analysis of dodecyl sulfate eluates of opsonized particles, cleansed of many contaminating proteins by boiling with 2 M NaCl (yet still opsonized), revealed that the polypeptide with C3-derived radioactivity had a mol wt of approximately 140,000 and was composed of 70,000 mol wt subunits linked by disulfide bonds. Immunochemical analysis and comparison of the peptide structure of the eluate with that of C3 indicated that the opsonic fragment is not the fragment defined as C3b but a smaller derivative of C3.

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