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Claudio Scazzocchio - One of the best experts on this subject based on the ideXlab platform.
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the nada gene of aspergillus nidulans encoding Adenine Deaminase is subject to a unique regulatory pattern
Fungal Genetics and Biology, 2008Co-Authors: Nathalie Oestreicher, Carin Ribard, Claudio ScazzocchioAbstract:The Adenine Deaminase of A. nidulans, encoded by nadA, can be considered both as a catabolic and a purine salvage enzyme. We show that its transcriptional regulation reflects this double metabolic role. As all other genes involved in purine utilisation it is induced by uric acid, and this induction is mediated by the UaY transcription factor. However, it is also independently and synergistically induced by adenosine by a UaY-independent mechanism. At variance with all other enzymes of purine catabolism it is not repressed but induced by ammonium. This is at least partly due to the ammonium responsive GATA factor, AreA, acting in the nadA promoter as a competitor rather than in synergy with UaY. The adB gene, encoding adenylo-succinate synthetase, which can be considered both a biosynthetic and a salvage pathway enzyme, shares with nadA both ammonium and adenosine induction.
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sub families of α β barrel enzymes a new Adenine Deaminase family
Journal of Molecular Biology, 2003Co-Authors: Carin Ribard, Michel Rochet, Bernard Labedan, Bertrand Daignanfornier, Pedro M Alzari, Claudio Scazzocchio, Nathalie OestreicherAbstract:Abstract No gene coding for an Adenine Deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that Adenine Deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the Adenine Deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A. nidulans gene was expressed in Escherichia coli and the purified enzyme shows Adenine but not adenosine Deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine Deaminases rather than to the bacterial Adenine Deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal Adenine Deaminases and the homologous adenosine Deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that Adenine Deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the α/β barrel scaffold.
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Sub-families of α/β barrel enzymes: A new Adenine Deaminase family
Journal of Molecular Biology, 2003Co-Authors: Carin Ribard, Michel Rochet, Bernard Labedan, Pedro M Alzari, Claudio Scazzocchio, Bertrand Daignan-fornier, Nathalie OestreicherAbstract:Abstract No gene coding for an Adenine Deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that Adenine Deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the Adenine Deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A. nidulans gene was expressed in Escherichia coli and the purified enzyme shows Adenine but not adenosine Deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine Deaminases rather than to the bacterial Adenine Deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal Adenine Deaminases and the homologous adenosine Deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that Adenine Deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the α/β barrel scaffold.
Bertrand Daignanfornier - One of the best experts on this subject based on the ideXlab platform.
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proteasome and scf dependent degradation of yeast Adenine Deaminase upon transition from proliferation to quiescence requires a new f box protein named saf1p
Molecular Microbiology, 2006Co-Authors: Stephanie Escusa, Jurgi Camblong, Jeanmarc Galan, Benoit Pinson, Bertrand DaignanfornierAbstract:Summary In response to nutrient limitation, Saccharomyces cerevisiae cells enter into a non-proliferating state termed quiescence. This transition is associated with profound changes in gene expression patterns. The Adenine Deaminase encoding gene AAH1 is among the most precociously and tightly downregulated gene upon entry into quiescence. We show that AAH1 downregulation is not specifically due to glucose exhaustion but is a more general response to nutrient limitation. We also found that Aah1p level is tightly correlated to RAS activity indicating thus an important role for the protein kinase A pathway in this regulation process. We have isolated three deletion mutants, srb10, srb11 and saf1 (ybr280c) affecting AAH1 expression during post-diauxic growth and in early stationary phase. We show that the Srb10p cyclin-dependent kinase and its cyclin, Srb11p, regulate AAH1 expression at the transcriptional level. By contrast, Saf1p, a previously uncharacterized F-box protein, acts at a post-transcriptional level by promoting degradation of Aah1p. This post-transcriptional regulation is abolished by mutations affecting the proteasome or constant subunits of the SCF (Skp1–Cullin–F-box) complex. We propose that Saf1p targets Aah1p for proteasome-dependent degradation upon entry into quiescence. This work provides the first direct evidence for active degradation of proteins in quiescent yeast cells.
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sub families of α β barrel enzymes a new Adenine Deaminase family
Journal of Molecular Biology, 2003Co-Authors: Carin Ribard, Michel Rochet, Bernard Labedan, Bertrand Daignanfornier, Pedro M Alzari, Claudio Scazzocchio, Nathalie OestreicherAbstract:Abstract No gene coding for an Adenine Deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that Adenine Deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the Adenine Deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A. nidulans gene was expressed in Escherichia coli and the purified enzyme shows Adenine but not adenosine Deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine Deaminases rather than to the bacterial Adenine Deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal Adenine Deaminases and the homologous adenosine Deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that Adenine Deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the α/β barrel scaffold.
Joan M. Hevel - One of the best experts on this subject based on the ideXlab platform.
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Assays for S-adenosylmethionine (AdoMet/SAM)-dependent methyltransferases.
Current protocols in toxicology, 2020Co-Authors: Whitney L. Wooderchak, Zhaohui Sunny Zhou, Joan M. HevelAbstract:Modification of small molecules and proteins by methyltransferases impacts a wide range of biological processes. Here we report two methods for measuring methyltransferase activity. First we describe an enzyme-coupled continuous spectrophotometric assay used to quantitatively characterize S-adenosyl-L-methionine (AdoMet or SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy or SAH), the transmethylation product of AdoMet-dependent methyltransferase, is hydrolyzed to S-ribohomocysteine and Adenine by recombinant AdoHcy nucleosidase. Subsequently, the Adenine generated from AdoHcy is further hydrolyzed to homoxanthine and ammonia by recombinant Adenine Deaminase. This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Secondly, we describe a discontinuous assay that follows radiolabel incorporation into the methyl receptor. An advantage of both assays is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Importantly both methods are inexpensive, robust, and amenable to high throughput.
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Current Protocols in Toxicology - Assays for S-adenosylmethionine (AdoMet/SAM)-dependent methyltransferases.
Current protocols in immunology, 2008Co-Authors: Whitney L. Wooderchak, Zhaohui Sunny Zhou, Joan M. HevelAbstract:Modification of small molecules and proteins by methyltransferases impacts a wide range of biological processes. Here we report two methods for measuring methyltransferase activity. First we describe an enzyme-coupled continuous spectrophotometric assay used to quantitatively characterize S-adenosyl-l-methionine (AdoMet or SAM)–dependent methyltransferase activity. In this assay, S-adenosyl-l-homocysteine (AdoHcy or SAH), the transmethylation product of AdoMet-dependent methyltransferase, is hydrolyzed to S-ribohomocysteine and Adenine by recombinant AdoHcy nucleosidase. Subsequently, the Adenine generated from AdoHcy is further hydrolyzed to homoxanthine and ammonia by recombinant Adenine Deaminase. This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Secondly, we describe a discontinuous assay that follows radiolabel incorporation into the methyl receptor. An advantage of both assays is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Importantly both methods are inexpensive, robust, and amenable to high throughput. Curr. Protoc. Toxicol. 38:4.26.1-4.26.12. © 2008 by John Wiley & Sons, Inc. Keywords: methyltransferase; SAM; AdoMet; S-adenosyl methionine; assays; SAH; AdoHcy nucleosidase; Adenine Deaminase; S-ribosylhomocysteine; Adenine
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An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases.
Analytical Biochemistry, 2006Co-Authors: Kathleen M. Dorgan, Whitney L. Wooderchak, Zhaohui Sunny Zhou, Donraphael P. Wynn, Erin L. Karschner, Joshua F. Alfaro, Joan M. HevelAbstract:Abstract Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S -adenosyl- l -methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S -adenosyl- l -homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S -ribosylhomocysteine and Adenine by recombinant S -adenosylhomocysteine/5′-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, Adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant Adenine Deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N -methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S -adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5′-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.
Narendra K Bairwa - One of the best experts on this subject based on the ideXlab platform.
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Genetic Interaction between RLM1 and F-box Motif Encoding gene SAF1 Contributes to Stress Response in Saccharomyces cerevisiae
bioRxiv, 2019Co-Authors: Meenu Sharma, V. Verma, Narendra K BairwaAbstract:Abstract Stress response is mediated by transcription of stress responsive genes. F-box motif protein Saf1 involves in SCF-E3 ligase mediated degradation of the Adenine Deaminase, Aah1 upon nutrient stress. Four transcription regulators, BUR6, MED6, SPT10, SUA7, have been reported for SAF1 gene in genome database of Saccharomyces cerevisiae. Here in this study an in-silco analysis of gene expression and transcription factor databases was carried out to understand the regulation of SAF1 gene expression during stress for hypothesis generation and experimental analysis. The GEO profile database analysis showed increased expression of SAF1 gene when treated with clioquinol, pterostilbene, gentamicin, hypoxia, genotoxic, desiccation, and heat stress, in WT cells. SAF1 gene expression in stress conditions correlated positively whereas AAH1 expression negatively with RLM1 transcription factor, which was not reported earlier. Based on analysis of expression profile and regulatory association of SAF1 and RLM1, we hypothesized that inactivation of both the genes may contribute to stress tolerance. The experimental analysis with the double mutant, saf1Δrlm1Δ for cellular growth response to stress causing agents, showed tolerance to calcofluor white, SDS, and hydrogen peroxide. On the contrary, saf1Δrlm1Δ showed sensitivity to MMS, HU, DMSO, Nocodazole, Benomyl stress. Based on in-silico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.
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Absence of Replication fork associated factor CTF4 and F-box motif Encoding Gene SAF1 leads to reduction in Cell Size and Stress Tolerance Phenotype in S. cerevisiae
bioRxiv, 2019Co-Authors: Meenu Sharma, Samar Singh, V. Verma, Narendra K BairwaAbstract:Chromosome transmission fidelity factor, Ctf4 in S. cerevisiae associates with replication fork and helps in the sister chromatid cohesion. At the replication fork, Ctf4 links DNA helicase with the DNA polymerase. The absence of Ctf4 invokes replication checkpoint in the cells. The Saf1 of S.cerevisiae interacts with Skp1 of SCF-E3 ligase though F box-motif and ubiquitinates the Adenine Deaminase Aah1 during phase transition due to nutrient stress. The genetic interaction between the CTF4 and SAF1 has not been studied. Here we report genetic interaction between CTF4 and SAF1 which impacts the growth fitness and response to stress. The single and double gene deletions of SAF1 and CTF4 were constructed in the BY4741 genetic background. The strains were tested for growth on rich media and media containing stress causing agents. The saf1{Delta}ctf4{Delta} cells with reduced cell size showed the fastest growth phenotype on YPD medium when compared with the saf1{Delta}, ctf4{Delta}, and WT. The saf1{Delta}ctf4{Delta} cells also showed the tolerance to MMS, NaCl, Glycerol, SDS, Calcofluor white, H2O2, DMSO, Benomyl, and Nocodazole when compared with the saf1{Delta}, ctf4{Delta}, and WT cells. However, saf1{Delta}ctf4{Delta} cells showed the sensitivity to HU when compared with WT and saf1{Delta}. Based on these observations we suggest that SAF1 and CTF4 interact genetically to regulate the cell size, growth and stress response.
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The absence of F-box motif Encoding Gene SAF1 and Chromatin Associated factor CTF8 together contributes to MMS Resistant and HU Sensitive phenotype in S. cerevisiae
bioRxiv, 2019Co-Authors: Meenu Sharma, V. Verma, Narendra K BairwaAbstract:Abstract The Replication factor-C compex which related to cohesion, constitutes, three subunits called Ctf18, Ctf8 and Dcc1. These three subunit complex assist the loading of PCNA onto the chromosome. None of the replication factor C components are essential for cell viability. The null mutant of the CTF8 in S.cerevisiae shows the chromosome instability and high frequency of chromosome loss. The SAF1 gene product of S. cerevisiae involved in the degradation of Adenine Deaminase factor Aah1p by SCF-E3 ligase, which itself is the part of E3 ligase. The ubiquitin marked degradation of Aah1p occurs during nutrient stress which lead to cell enter into the quiescent state. The N-terminus of Saf1p interacts with the Skp1 of SCF-E3 ligase and at C-terminus recruits with Aah1p. Here we have investigated about the binary genetic interaction between the SAF1 and CTF8 genes. The strains containing single and double gene deletions of SAF1 and CTF8 were constructed in the BY4741 genetic background. Further the mutant strains were evaluated for growth fitness, genome stability and response to genotoxic stress caused by hydroxyurea (HU) and methyl methane sulfonate (MMS). The saf1Δctf8Δ strain showed the increased growth phenotype in comparison to saf1Δ, ctf8Δ, and WT strain on YPD medium. However saf1Δctf8Δ strain when grown in the presence MMS showed resistance and HU sensitive phenotype when compared with saf1Δ, ctf8Δ. The frequency of Ty1 retro-transposition was also elevated in saf1Δctf8Δ in comparison to either saf1Δ or ctf8Δ. The number of cells showing the two or multi-nuclei phenotype was also increased in saf1Δctf8Δ cells when compared with the either saf1Δ or ctf8Δ. Based on these observations, we report that the absence of both the gene SAF1 and CTF8 together leads to MMS resistance, HU sensitivity, and genome instability. This report warrants the investigation of mechanisms of differential growth phenotype due to loss of SAF1 and CTF8 together in presence of genotoxic stress in future.
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Loss of F-box Motif Encoding Gene SAF1 and RRM3 Together Leads to Synthetic Growth Defect and Sensitivity to HU, MMS in S.cerevisiae
bioRxiv, 2019Co-Authors: Meenu Sharma, V. Verma, Narendra K BairwaAbstract:Unearthing of novel genetic interaction which leads to synthetic growth defects due to inactivation of genes are needed for applications in precision medicine. The genetic interactions among the molecular players involving different biological pathways need to be investigated. The SAF1 gene of S.cerevisiae encodes for a protein product which contain N-terminal F-box motif and C-terminal RCC1 domain. The F-box motif interacts with Skp1subunit of the SCF-E3 ligase and C-terminus with Aah1 (Adenine Deaminase) for ubiquitination and subsequent degradation by 26S proteasome during phase transition from proliferation state to quiescence phase due to nutrient limitation stress. The replication fork associated protein Rrm3 of S.cerevisiae belongs to Pif1 family helicase and function in removal of the non-histone proteins during replication fork movement. Here we have investigated the genetic interaction among both the genes (SAF1 and RRM3) and their role in growth fitness and genome stability. The single and double gene knockout strains of SAF1and RRM3 genes was constructed in BY4741 genetic background and checked for the growth fitness in presence of genotoxic stress causing agents such as hydroxyurea and methyl methanesulfonate. The strains were also evaluated for nuclear migration defect by DAPI staining and for HIS3AI marked Ty1 retro-transposition. The saf1{Delta}rrm3{Delta} showed the extremely slow growth phenotype in rich medium and sensitivity to genotoxic agents such as HU and MMS in comparison to single gene mutant (saf1{Delta}, rrm3{Delta}) and WT cells. The saf1{Delta}rrm3{Delta} also showed the defects in nuclear migration as evident by multi-nuclei phenotype. The saf1{Delta}rrm3{Delta} also showed the elevated frequency of Ty1 retro-transposition in JC2326 background in comparison to either saf1{Delta} or rrm3{Delta}. Based on these observations we report that thatSAF1 and RRM3 functions in parallel pathway for growth fitness and stability of the genome.
Nathalie Oestreicher - One of the best experts on this subject based on the ideXlab platform.
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the nada gene of aspergillus nidulans encoding Adenine Deaminase is subject to a unique regulatory pattern
Fungal Genetics and Biology, 2008Co-Authors: Nathalie Oestreicher, Carin Ribard, Claudio ScazzocchioAbstract:The Adenine Deaminase of A. nidulans, encoded by nadA, can be considered both as a catabolic and a purine salvage enzyme. We show that its transcriptional regulation reflects this double metabolic role. As all other genes involved in purine utilisation it is induced by uric acid, and this induction is mediated by the UaY transcription factor. However, it is also independently and synergistically induced by adenosine by a UaY-independent mechanism. At variance with all other enzymes of purine catabolism it is not repressed but induced by ammonium. This is at least partly due to the ammonium responsive GATA factor, AreA, acting in the nadA promoter as a competitor rather than in synergy with UaY. The adB gene, encoding adenylo-succinate synthetase, which can be considered both a biosynthetic and a salvage pathway enzyme, shares with nadA both ammonium and adenosine induction.
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sub families of α β barrel enzymes a new Adenine Deaminase family
Journal of Molecular Biology, 2003Co-Authors: Carin Ribard, Michel Rochet, Bernard Labedan, Bertrand Daignanfornier, Pedro M Alzari, Claudio Scazzocchio, Nathalie OestreicherAbstract:Abstract No gene coding for an Adenine Deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that Adenine Deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the Adenine Deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A. nidulans gene was expressed in Escherichia coli and the purified enzyme shows Adenine but not adenosine Deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine Deaminases rather than to the bacterial Adenine Deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal Adenine Deaminases and the homologous adenosine Deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that Adenine Deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the α/β barrel scaffold.
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Sub-families of α/β barrel enzymes: A new Adenine Deaminase family
Journal of Molecular Biology, 2003Co-Authors: Carin Ribard, Michel Rochet, Bernard Labedan, Pedro M Alzari, Claudio Scazzocchio, Bertrand Daignan-fornier, Nathalie OestreicherAbstract:Abstract No gene coding for an Adenine Deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that Adenine Deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the Adenine Deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A. nidulans gene was expressed in Escherichia coli and the purified enzyme shows Adenine but not adenosine Deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine Deaminases rather than to the bacterial Adenine Deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal Adenine Deaminases and the homologous adenosine Deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that Adenine Deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the α/β barrel scaffold.
