scholarly journals A novel allele of HAP1 causes uninducible expression of HEM13 in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 136 (3) ◽  
pp. 819-831 ◽  
Author(s):  
S C Ushinsky ◽  
T Keng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Mutant Allele ◽  
Binding Activity ◽  
The Other ◽  
Wild Type ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Allele Expression ◽  
Novel Allele

Abstract Transcription of HEM13 in Saccharomyces cerevisiae is repressed by heme and oxygen. We have isolated two mutants in which expression of HEM13 is aberrant. The mutant alleles in these strains represent two different alleles of HAP1. HAP1 encodes an activator protein whose DNA binding activity is stimulated by heme, and is required for the transcription of CYC1, ROX1 and a number of other heme-dependent genes. One of our mutant alleles confers a phenotype much like that of the hap1::LEU2 allele. Expression of HEM13 in a strain with this mutation is elevated under repressing conditions and not fully inducible in the absence of heme. The other mutant allele of HAP1 we uncovered confers a novel phenotype. A strain containing this allele exhibits heme-independent expression of CYC1 and ROX1 and uninducible expression of HEM13 and ANB1. The mutation associated with this novel allele of HAP1 was localized to a glycine to aspartate change in amino acid 235 of HAP1, between the DNA binding and heme responsive domains. DNA binding assays demonstrated that the protein made from this HAP1 allele retains the ability to bind DNA, but that unlike wild-type HAP1 protein, this binding is not stimulated by heme.

scholarly journals E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

1990 ◽  
Vol 10 (10) ◽  
pp. 5138-5149 ◽  
Author(s):  
R J Rooney ◽  
P Raychaudhuri ◽  
J R Nevins
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Stable Complex ◽  
Small Subset ◽  
Adenovirus Infection ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Sequence Recognition ◽  
Nuclear Factors ◽  
Dna Binding Factors

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

Analysis Of E2A Point Mutations In B Cell Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4850-4850
Author(s):  
Eroica Soans ◽  
John K Choi
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Binding Activity ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Cell Leukemia ◽  
Wild Type ◽  
Dna Binding Activity ◽  
E Box ◽  
B Cell Leukemia

Abstract Introduction TCF3 encodes for E2A protein, which belongs to the helix loop helix transcription factor family. E2A activates transcription of downstream genes by binding to E-box motifs as a homo or hetero dimer. E2A plays an important role in B lymphocyte development. Therefore deletion or mutations in TCF3 or even lowered activity of E2A are causes of B cell leukemia and lymphomas. Recently, three mutations V557E, D561E and N551K in E2A were isolated in Burkitt’s lymphoma (Schmitz, Young et al. 2012). The first two mutations are present in the homo dimerization region of E2A while N551K is present in the DNA binding region. Though the paper enumerated role of TCF3 in Burkitt’s lymphoma but the significance of these TCF3 mutations or mechanism needed further characterization. We hypothesized that these TCF3 mutations have an alternate mechanism as compared to wild type TCF3 and therefore may affect B cell development. Methods We characterized three TCF3 mutants by cloning them into in MIGR1 backbone using TOPO cloning. E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells. DNA binding activity was measured using a DNA protein binding colorimetric assay. Results V557E and D561E mutants have lower activity as compared to wild type E2A as studied using E2A-specific luciferase reporter assay; while N551K showed no activity in the same assay as compared to wild type E2A activity. Similarly V557E and D561 form weaker bonds with the E box motifs while N551K showed no DNA binding activity as studied using colorimetric DNA-protein binding assay. The plasmid expressions were verified using western blot analysis. Conclusion Our findings suggest mutations V557E and D561E may follow a similar pathway as wild-type E2A but have lower activity. The N551K mutation has an alternate pathway to wild type TCF3 that may impact B cell proliferation, survival and development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Agnieszka Siomek ◽  
Kamil Brzoska ◽  
Barbara Sochanowicz ◽  
Daniel Gackowski ◽  
Rafal Rozalski ◽  
...  
Keyword(s):  
Dna Damage ◽  
Superoxide Dismutase ◽  
Dna Binding ◽  
Urinary Excretion ◽  
Oxidative Dna Damage ◽  
Binding Activity ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Organ Specific ◽  
Damaged Dna

Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites

1990 ◽  
Vol 10 (2) ◽  
pp. 859-862
Author(s):  
G M Santangelo ◽  
J Tornow
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Heterologous Gene ◽  
Dna Binding Activity ◽  
Glycolytic Gene

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences

1989 ◽  
Vol 9 (7) ◽  
pp. 2906-2913
Author(s):  
S C Francesconi ◽  
S Eisenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Binding Activity ◽  
Physical Parameters ◽  
Major Protein ◽  
Dna Binding Activity ◽  
Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

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