scholarly journals RPH1 and GIS1 Are Damage-Responsive Repressors of PHR1

1999 ◽  
Vol 19 (11) ◽  
pp. 7630-7638 ◽  
Author(s):  
Yeun Kyu Jang ◽  
Ling Wang ◽  
Gwendolyn B. Sancar
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Protein S ◽  
Repair Gene ◽  
Repair Capacity ◽  
Pyrimidine Dimers ◽  
Dna Repair Gene ◽  
Damage Response

ABSTRACT The Saccharomyces cerevisiae DNA repair genePHR1 encodes a photolyase that catalyzes the light-dependent repair of pyrimidine dimers. PHR1expression is induced at the level of transcription by a variety of DNA-damaging agents. The primary regulator of the PHR1damage response is a 39-bp sequence called URS PHR1 which is the binding site for a protein(s) that constitutes the damage-responsive repressor PRP. In this communication, we report the identification of two proteins, Rph1p and Gis1p, that regulate PHR1 expression through URS PHR1 . Both proteins contain two putative zinc fingers that are identical throughout the DNA binding region, and deletion of both RPH1 and GIS1 is required to fully derepress PHR1 in the absence of damage. Derepression of PHR1 increases the rate and extent of photoreactivation in vivo, demonstrating that the damage response of PHR1enhances cellular repair capacity. In vitro footprinting and binding competition studies indicate that the sequence AG4(C4T) within URS PHR1 is the binding site for Rph1p and Gis1p and suggests that at least one additional DNA binding component is present in the PRP complex.

scholarly journals Multiple Functions of the Nonconserved N-Terminal Domain of Yeast TATA-Binding Protein

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 87-93
Author(s):  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Protein S ◽  
Tata Binding Protein ◽  
Terminal Region ◽  
Core Domain ◽  
Terminal Domain ◽  
Two Factors

Abstract The TATA-binding protein (TBP) is composed of a highly conserved core domain sufficient for TATA-element binding and preinitiation complex formation as well as a highly divergent N-terminal region that is dispensable for yeast cell viability. In vitro, removal of the N-terminal region domain enhances TBP-TATA association and TBP dimerization. Here, we examine the effects of truncation of the N-terminal region in the context of yeast TBP mutants with specific defects in DNA binding and in interactions with various proteins. For a subset of mutations that disrupt DNA binding and the response to transcriptional activators, removal of the N-terminal domain rescues their transcriptional defects. By contrast, deletion of the N-terminal region is lethal in combination with mutations on a limited surface of TBP. Although this surface is important for interactions with TFIIA and Brf1, TBP interactions with these two factors do not appear to be responsible for this dependence on the N-terminal region. Our results suggest that the N-terminal region of TBP has at least two distinct functions in vivo. It inhibits the interaction of TBP with TATA elements, and it acts positively in combination with a specific region of the TBP core domain that presumably interacts with another protein(s).

In vivo stimulation of a chimeric promoter by binding sites for nuclear factor I

1991 ◽  
Vol 11 (6) ◽  
pp. 2946-2951
Author(s):  
J J Knox ◽  
P J Rebstein ◽  
A Manoukian ◽  
R M Gronostajski
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Tata Box ◽  
Nuclear Factor ◽  
Chimeric Promoter ◽  
Factor I ◽  
Nuclear Factor I

Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box.

scholarly journals A Protein in the Yeast Saccharomyces cerevisiae Presents DNA Binding Homology to the p53 Checkpoint Protein and Tumor Suppressor

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 417
Author(s):  
Kanwal Farooqi ◽  
Marjan Ghazvini ◽  
Leah D. Pride ◽  
Louis Mazzella ◽  
David White ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Site ◽  
Consensus Sequence ◽  
Protein S ◽  
Mobility Shift ◽  
Yeast Saccharomyces Cerevisiae ◽  
In Vivo Test ◽  
Site Factor

Saccharomyces cerevisiae does not contain a p53 homolog. Utilizing this yeast as an in vivo test tube model, our aim was to investigate if a yeast protein would show p53 DNA binding homology. Electrophoretic mobility shift analyses revealed the formation of specific DNA-protein complexes consisting of S. cerevisiae nuclear protein(s) and oligonucleotides containing p53 DNA binding sites. A S. cerevisiae p53 binding site factor (Scp53BSF) bound to a p53 synthetic DNA-consensus sequence (SCS) and a p53 binding-site sequence from the MDM2 oncogene. The complexes were of comparable size. Like mammalian p53, the affinity of Scp53BSF for the SCS oligonucleotide was higher than for the MDM2 oligonucleotide. Binding of Scp53BSF to the SCS and MDM2 oligonucleotides was strongly competed by unlabeled oligonucleotides containing mammalian p53 sites, but very little by a mutated site oligonucleotide. Importantly, Scp53BSF-DNA binding activity was significantly induced in extracts from cells with DNA damage. This resulted in dose-dependent coordinated activation of transcription when using p53-binding site reporter constructs. An ancient p53-like DNA binding protein may have been found, and activation of DNA-associated factors to p53 response elements may have functions not yet determined.

scholarly journals Metabolism and biochemical properties of nicotinamide adenine dinucleotide (NAD) analogs, nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Keisuke Yaku ◽  
Keisuke Okabe ◽  
Maryam Gulshan ◽  
Kiyoshi Takatsu ◽  
Hiroshi Okamoto ◽  
...  
Keyword(s):  
Stress Responses ◽  
Nicotinamide Adenine Dinucleotide ◽  
Biochemical Properties ◽  
Nicotinamide Adenine ◽  
Repair Gene ◽  
Nad Metabolism ◽  
Nad Glycohydrolase ◽  
Dna Repair Gene

Abstract Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that regulates various metabolic pathways, including glycolysis, β-oxidation, and oxidative phosphorylation. Additionally, NAD serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuin, and NAD glycohydrolase, and it regulates DNA repair, gene expression, energy metabolism, and stress responses. Many studies have demonstrated that NAD metabolism is deeply involved in aging and aging-related diseases. Previously, we demonstrated that nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD), which are analogs of NAD, are significantly increased in Nmnat3-overexpressing mice. However, there is insufficient knowledge about NGD and NHD in vivo. In the present study, we aimed to investigate the metabolism and biochemical properties of these NAD analogs. We demonstrated that endogenous NGD and NHD were found in various murine tissues, and their synthesis and degradation partially rely on Nmnat3 and CD38. We have also shown that NGD and NHD serve as coenzymes for alcohol dehydrogenase (ADH) in vitro, although their affinity is much lower than that of NAD. On the other hand, NGD and NHD cannot be used as substrates for SIRT1, SIRT3, and PARP1. These results reveal the basic metabolism of NGD and NHD and also highlight their biological function as coenzymes.

scholarly journals Assembly of a Functional Beta Interferon Enhanceosome Is Dependent on ATF-2–c-jun Heterodimer Orientation

2000 ◽  
Vol 20 (13) ◽  
pp. 4814-4825 ◽  
Author(s):  
James V. Falvo ◽  
Bhavin S. Parekh ◽  
Charles H. Lin ◽  
Ernest Fraenkel ◽  
Tom Maniatis
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Site ◽  
Leucine Zipper ◽  
Critical Role ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Beta Interferon

ABSTRACT Heterodimeric transcription factors, including the basic region-leucine zipper (bZIP) protein ATF-2–c-jun, are well-characterized components of an enhanceosome that mediates virus induction of the human beta interferon (IFN-β) gene. Here we report that within the IFN-β enhanceosome the ATF-2–c-jun heterodimer binds in a specific orientation, which is required for assembly of a complex between ATF-2–c-jun and interferon regulatory factor 3 (IRF-3). We demonstrate that correct orientation of the ATF-2–c-jun binding site is required for virus induction of the IFN-β gene and for IRF-3-dependent activation of a composite ATF-2– c-jun–IRF site in the IFN-β promoter. We also show that in vitro the DNA-bound ATF-2–c-jun heterodimer adopts a fixed orientation upon the binding of IRF-3 at an adjacent site in the IFN-β enhancer and that the DNA-binding domain of IRF-3 is sufficient to mediate this effect. In addition, we show that the DNA-binding domain of ATF-2 is necessary and sufficient for selective protein-protein interactions with IRF-3. Strikingly, in vivo chromatin immunoprecipitation experiments with IFN-β reporter constructs reveal that recruitment of IRF-3 to the IFN-β promoter upon virus infection is dependent on the orientation of the ATF-2–c-jun heterodimer binding site. These observations demonstrate functional and physical cooperativity between the bZIP and IRF transcription factor families and illustrate the critical role of heterodimeric transcription factors in formation of the IFN-β enhanceosome.

RirA of Dinoroseobacter shibae senses iron via a [3Fe–4S]1+ cluster co-ordinated by three cysteine residues

2020 ◽  
Vol 477 (1) ◽  
pp. 191-212
Author(s):  
Maren Behringer ◽  
Lisa Plötzky ◽  
Dirk Baabe ◽  
Marc-Kevin Zaretzke ◽  
Peter Schweyen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Growth Conditions ◽  
Cysteine Residues ◽  
Mobility Shift ◽  
Paramagnetic Resonance ◽  
Dinoroseobacter Shibae ◽  
Electro Mobility

In the marine bacterium, Dinoroseobacter shibae the transcription factor rhizobial iron regulator A (RirA) is involved in the adaptation to iron-limited growth conditions. In vitro iron and sulfide content determinations in combination with UV/Vis and electron paramagnetic resonance (EPR) spectroscopic analyses using anaerobically purified, recombinant RirA protein suggested a [3Fe–4S]1+ cluster as a cofactor. In vivo Mössbauer spectroscopy also corroborated the presence of a [3Fe–4S]1+ cluster in RirA. Moreover, the cluster was found to be redox stable. Three out of four highly conserved cysteine residues of RirA (Cys 91, Cys 99, Cys 105) were found essential for the [3Fe–4S]1+ cluster coordination. The dimeric structure of the RirA protein was independent of the presence of the [3Fe–4S]1+ cluster. Electro mobility shift assays demonstrated the essential role of an intact [3Fe–4S]1+ cluster for promoter binding by RirA. The DNA binding site was identified by DNase I footprinting. Mutagenesis studies in combination with DNA binding assays confirmed the promoter binding site as 3′-TTAAN10AATT-5′. This work describes a novel mechanism for the direct sensing of cellular iron levels in bacteria by an iron-responsive transcriptional regulator using the integrity of a redox-inactive [3Fe–4S]1+ cluster, and further contributes to the general understanding of iron regulation in marine bacteria.

scholarly journals Sequence-Specific DNA Binding by the αNAC Coactivator Is Required for Potentiation of c-Jun-Dependent Transcription of the Osteocalcin Gene

2005 ◽  
Vol 25 (9) ◽  
pp. 3452-3460 ◽  
Author(s):  
Omar Akhouayri ◽  
Isabelle Quélo ◽  
René St-Arnaud
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Gene Transcription ◽  
Osteoblastic Differentiation ◽  
Proximal Promoter ◽  
Osteoblastic Cells ◽  
Specific Marker ◽  
Osteocalcin Gene

ABSTRACT Since the c-Jun coactivator αNAC was initially identified in a differential screen for genes expressed in differentiated osteoblasts, we examined whether the osteocalcin gene, a specific marker of terminal osteoblastic differentiation, could be a natural target for the coactivating function of αNAC. We had also previously shown that αNAC can specifically bind DNA in vitro, but it remained unclear whether the DNA-binding function of αNAC is expressed in vivo or if it is required for coactivation. We have identified an αNAC binding site within the murine osteocalcin gene proximal promoter region and demonstrated that recombinant αNAC or αNAC from ROS17/2.8 nuclear extracts can specifically bind this element. Using transient transfection assays, we have shown that αNAC specifically potentiated the c-Jun-dependent transcription of the osteocalcin promoter and that this activity specifically required the DNA-binding domain of αNAC. Chromatin immunoprecipitation confirmed that αNAC occupies its binding site on the osteocalcin promoter in living osteoblastic cells expressing osteocalcin. Inhibition of the expression of endogenous αNAC in osteoblastic cells by use of RNA interference provoked a decrease in osteocalcin gene transcription. Our results show that the osteocalcin gene is a target for the αNAC coactivating function, and we propose that αNAC is specifically targeted to the osteocalcin promoter through its DNA-binding activity as a means to achieve increased specificity in gene transcription.

scholarly journals Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ fromSchizosaccharomyces pombe

2000 ◽  
Vol 20 (23) ◽  
pp. 8958-8968 ◽  
Author(s):  
Young Sam Shim ◽  
Yeun Kyu Jang ◽  
Myung Sil Lim ◽  
Jung Sup Lee ◽  
Rho Hyun Seong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Repair Gene ◽  
Repair Capacity ◽  
Dna Damaging Agents ◽  
Dna Repair Gene ◽  
Damage Response

ABSTRACT The Schizosaccharomyces pombe DNA repair generhp51 + encodes a RecA-like protein with the DNA-dependent ATPase activity required for homologous recombination. The level of the rhp51 + transcript is increased by a variety of DNA-damaging agents. Its promoter has twocis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility. Here we report identification of Rdp1, which regulates rhp51 + expression through the DRE of rhp51 +. The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively. In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1. Mutations of both DRE1 and DRE2 affected the damage-induced expression ofrhp51 +, indicating that both DREs are required for transcriptional activation. In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response ofrhp51 + enhances the cellular repair capacity. Surprisingly, haploid cells containing a complete rdp1deletion could not be recovered, indicating thatrdp1 + is essential for cell viability and implying the existence of other target genes. Furthermore, the DNA damage-dependent expression of rhp51 + was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription ofrhp51 +.

scholarly journals Identification and Functional Characterization of a High-Affinity Bel-1 DNA Binding Site Located in the Human Foamy Virus Internal Promoter

1998 ◽  
Vol 72 (1) ◽  
pp. 504-511 ◽  
Author(s):  
Yibin Kang ◽  
Wade S. Blair ◽  
Bryan R. Cullen
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Foamy Virus ◽  
Promoter Element ◽  
Internal Promoter ◽  
Dna Binding Site ◽  
Foamy Viruses ◽  
Ltr Promoter

ABSTRACT The transcription of genes carried by primate foamy viruses is dependent on two distinct promoter elements. These are the long terminal repeat (LTR) promoter, which regulates expression of the viral structural proteins, and a second internal promoter, located towards the 3′ end of the env gene, that directs expression of the viral auxiliary proteins. One of these auxiliary proteins is a potent transcriptional transactivator, termed Bel-1 in human foamy virus (HFV) and Tas or Taf in the related simian foamy viruses, that is critical for foamy virus replication. Previously, it has been demonstrated that the LTR promoter element of HFV contains a DNA binding site for Bel-1 that is critical for transcriptional activation (F. He, W. S. Blair, J. Fukushima, and B. R. Cullen, J. Virol. 70:3902–3908, 1996). Here, we extended this earlier work by using methylation interference analysis to identify and characterize the Bel-1 DNA binding sites located in the HFV LTR and internal promoter elements. Based on these data, we propose a minimal, 25-bp DNA binding site for Bel-1, derived from the HFV internal promoter element, and show that this short DNA sequence mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrate that, as determined by both in vitro and in vivo assays, the Bel-1 target site located within the HFV internal promoter binds Bel-1 with a significantly higher affinity than the cap-proximal Bel-1 target site located in the LTR promoter. This result may provide a mechanistic explanation for the observation that the internal promoter is activated significantly earlier than the LTR promoter during the foamy virus life cycle.

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