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PubMedDate not supplied

Rat seminal-vesicle secretory protein SVS II binds DNA with a preference for the 5' regulatory region of secretory protein SVS IV gene: co-isolation with components of the nuclear matrix.

In rats, the ventral prostate and seminal vesicles produce distinct sets of proteins whose functions and tissue-specific regulation by androgens remain unclear. We have utilized the genes encoding the major secretory protein of seminal vesicles, SVS IV, and the C3 subunit of prostatein of the ventral prostate to study how the nuclear matrix might determine their tissue-specific gene expression. Nuclear matrix proteins were prepared from purified nuclei with DNase and 2 M NaCl, separated in SDS gels, and transferred onto membranes for DNA-binding (southwestern) and immunological (western) analyses. The 5' region of the SVS IV gene (SVS IV-7S) bound to a 45,000-kDa molecular-weight protein band in the nuclear matrix of seminal vesicles but not to that of ventral prostate, kidney, or liver. Sequencing revealed that this band was a seminal-vesicle secretory protein, SVS II, whose identity was confirmed with an anti-SVS II antiserum in western blots. Actin-like protein, similar in mobility to SVS II, was detected in seminal-vesicle and ventral prostate nuclear matrix, but not in seminal-vesicle fluid. Reducing agent (10 mM dithiothreitol) and acidic (pH 6.5) buffer did not eliminate SVS II, but isolation of nuclear matrices with ammonium sulfate, nucleases, and urea decreased SVS II immunoreactivity and removed actin-like protein. SVS II binding to SVS IV-7S DNA was greater than its binding to either a comparable fragment of the C3 gene or linearized pUC-19 plasmid, and it was not eliminated by a 100-fold competition. When seminal-vesicle fluid was mixed with rat liver, some SVS II co-isolated with the nuclear-matrix proteins, indicating that nonspecific interactions contribute to its association with the nucleoskeleton. However, these interactions may not represent the intracellular behavior of SVS II in seminal-vesicle epithelium. Sequence comparisons indicate significant homologies between SVS II and some other seminal proteins, including bovine caltrin, which, under the name seminalplasmin, is known to possess antimicrobial activity. Collectively, these data suggest that in addition to its known functions, SVS II may also bind extraneous DNA in seminal fluid. Additionally, SVS II may participate as a structural component in the organization of a tissue-specific seminal-vesicle nuclear matrix.

Animals↗

PubMed2002

Low intracellular zinc induces oxidative DNA damage, disrupts p53, NFkappa B, and AP1 DNA binding, and affects DNA repair in a rat glioma cell line.

Approximately 10% of the U.S. population ingests <50% of the current recommended daily allowance for zinc. We investigate the effect of zinc deficiency on DNA damage, expression of DNA-repair enzymes, and downstream signaling events in a cell-culture model. Low zinc inhibited cell growth of rat glioma C6 cells and increased oxidative stress. Low intracellular zinc increased DNA single-strand breaks (comet assay). Zinc-deficient C6 cells also exhibited an increase in the expression of the zinc-containing DNA-repair proteins p53 and apurinic endonuclease (APE). Repletion with zinc restored cell growth and reversed DNA damage. APE is a multifunctional protein that not only repairs DNA but also controls DNA-binding activity of many transcription factors that may be involved in cancer progression. The ability of the transcription factors p53, nuclear factor kappaB, and activator protein 1 (AP1) to bind to consensus DNA sequences was decreased markedly with zinc deficiency, as assayed by electrophoretic mobility-shift assays. Thus, low intracellular zinc status causes oxidative DNA damage and induces DNA-repair protein expression, but binding of p53 and important downstream signals leading to proper DNA repair are lost without zinc.

Animals↗

PubMed1996

Identification of a novel domain in the aryl hydrocarbon receptor required for DNA binding.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the AHR nuclear translocator protein (ARNT). Both proteins possess basic helix-loop-helix motifs. ARNT binds to the side of the xenobiotic responsive element (XRE) that resembles an E-box (the sequence recognized by the majority of other basic helix-loop-helix proteins), whereas AHR binds to the side of the XRE that does not conform to the E-box sequence. The basic region of ARNT closely resembles those of other E-box-binding proteins, whereas the "nominal basic region" of AHR (amino acids 27 39), although required for XRE binding, deviates from this consensus. By extensive mutational analysis it is shown here that an additional block of amino acids of AHR (from tyrosine 9 to lysine 20) that contains a highly basic segment is required for XRE binding and transcriptional activation. Deletion of the first nine amino acids negates XRE binding. Substitution of either tyrosine 9 or arginine 14 with alanine eliminates XRE binding, whereas alanine substitutions at certain other sites within the block reduce but do not eliminate binding. The reported absence of the first nine amino acids in the purified protein may therefore be artifactual. These results suggest that the amino acids of AHR involved in binding to the XRE constitute a novel DNA-binding domain, comprising amino acids located within and amino-terminal to the nominal basic region.

Alanine↗

PubMed1996

Granulocytic protein p25 is a DNA-binding subunit of protein M(r) = 50,000: subcellular localization, cell and species specificity.

We have previously reported the presence and isolation of the novel protein M(r) = 25,000 (p25) from human granulocytes. In this study, the protein p25 was characterized by its: (a) ability to bind DNA, (b) subunit association, (c) partial protein sequencing, (d) subcellular localization, (e) cellular and species specificity and (f) stability in the presence of released granulocytic proteinases. For the detection of p25 in various extracts, fractions and types of human or animal hematopoietic cells, SDS-PAGE/Western blotting and immunohistochemical staining were used. The protein p25 was subjected to N-terminal amino acid sequence analysis. Protein p25-DNA interactions were monitored using Southwestern blotting. Selective inhibition of granulocytic proteinases was performed. Granulocytic protein p25 was found to be a product of oxidative cleavage of disulfide bridges in the p50 dimer. It was shown that neither protein p50 nor the p25 subunit is a degradation product of a protein of higher molecular weight. The N-terminal amino acid sequence of p25 was: RLNYNKPHAA. Binding capacity for double stranded DNA without significant sequence specificity was revealed and nuclear localization of some fraction of p50 dimer was established. The data concerning the cell and species specificity demonstrated that the protein is expressed only in normal human granulocytes. In summary, protein p25 originates from splitting of the p50 dimer. This subunit shows no identity with proteins already sequenced. DNA-binding of p25 is not sequence specific. It is concluded that the protein p50 is localized in the nuclei and cytoplasmic granules of mature human polymorphonuclear leukocytes or granulocytes of species high on the evolutionary tree. The functions of this protein remain to be determined.

Amino Acid Sequence↗

PubMed2004

Genetic and functional analyses of the øX174 DNA binding protein: the effects of substitutions for amino acid residues that spatially organize the two DNA binding domains.

The øX174 DNA binding protein contains two DNA binding domains, containing a series of DNA binding basic amino acids, separated by a proline-rich linker region. Within each DNA binding domain, there is a conserved glycine residue. Glycine and proline residues were mutated and the effects on virion structure were examined. Substitutions for glycine residues yield particles with similar properties to previously characterized mutants with substitutions for DNA binding residues. Both sets of mutations share a common extragenic second-site suppressor, suggesting that the defects caused by the mutant proteins are mechanistically similar. Hence, glycine residues may optimize DNA-protein contacts. The defects conferred by substitutions for proline residues appear to be fundamentally different. The properties of the mutant particles along with the atomic structure of the virion suggest that the proline residues may act to guide the packaged DNA to the adjacent fivefold related asymmetric unit, thus preventing a chaotic packaging arrangement.

Amino Acid Sequence↗

PubMed1998

Alterations in the GAL4 DNA-binding domain can affect transcriptional activation independent of DNA binding.

The GAL4 protein belongs to a large class of fungal transcriptional activator proteins encoding within their DNA-binding domains (DBD) six cysteines that coordinate two atoms of zinc (the Zn2Cys6 domain). In an effort to characterize the interactions between the Zn2Cys6 class transcriptional activator proteins and their DNA-binding sites, we have replaced in the full-length GAL4 protein small regions of the Zn2Cys6 domain with the analogous regions of another Zn2Cys6 protein called PPR1 an activator of pyrimidine biosynthetic genes. Alterations between the first and third cysteines abolished binding to GAL4 (upstream activation sequence of GAL (UASG)) or PPR1 (upstream acitvation sequence of UAS) DNA-binding sites and severely reduced transcriptional activation in yeast. In contrast, alterations between the third and fourth cysteines had only minor effects on binding to UASG but led to substantial decreases in activation in both yeast and a mammalian cell line. In the crystal structure of the GAL4 DBD-UASG complex (Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S. C. (1992) Nature 356, 408-414), this region is facing away from the DNA, making it likely that there exists within the GAL4 DBD an accessible domain important in activation.

Amino Acid Sequence↗

PubMed2004

Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding.

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.

Amino Acid Sequence↗

PubMed1997

A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor.

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.

Actins↗

PubMed2002

Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity.

DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.

Amino Acid Sequence↗

PubMed1997

Reversible activation of mouse metal response element-binding transcription factor 1 DNA binding involves zinc interaction with the zinc finger domain.

The DNA-binding activity of the Zn finger protein metal response element-binding transcription factor 1 (MTF-1) was rapidly induced both in vivo in mouse Hepa cells, canine MDCK, and human HeLa cells after incubation in medium containing zinc and in vitro in whole-cell extracts to which zinc was added. Acquisition of DNA-binding capacity in the presence of free zinc was temperature and time dependent and did not occur at 4 degrees C. In contrast, activated MTF-1 binding to the metal response element occurred at 4 degrees C. After Zn activation, mouse MTF-1 binding activity was more sensitive to EDTA and was stabilized by DNA binding relative to the Zn finger transcription factor Sp1. After dilution of nuclear or whole-cell extracts from Zn-treated cells and incubation at 37 degrees C, mouse MTF-1 DNA-binding activity was no longer detected but could be completely reconstituted by the subsequent readdition of zinc. In vitro-synthesized, recombinant mouse MTF-1 displayed a similar, reversible temperature- and Zn-dependent activation of DNA-binding activity. Analysis of deletion mutants of recombinant MTF-1 suggests that the Zn finger domain is important for the Zn-dependent activation of DNA-binding capacity. Thus, mouse MTF-1 functions as a reversibly activated sensor of free zinc pools in the cell.

Animals↗

PubMed2003

The DNA-binding domain of human papillomavirus type 18 E1. Crystal structure, dimerization, and DNA binding.

High risk types of human papillomavirus, such as type 18 (HPV-18), cause cervical carcinoma, one of the most frequent causes of cancer death in women worldwide. DNA replication is one of the central processes in viral maintenance, and the machinery involved is an excellent target for the design of antiviral therapy. The papillomaviral DNA replication initiation protein E1 has origin recognition and ATP-dependent DNA melting and helicase activities, and it consists of a DNA-binding domain and an ATPase/helicase domain. While monomeric in solution, E1 binds DNA as a dimer. Dimerization occurs via an interaction of hydrophobic residues on a single alpha-helix of each monomer. Here we present the crystal structure of the monomeric HPV-18 E1 DNA-binding domain refined to 1.8-A resolution. The structure reveals that the dimerization helix is significantly different from that of bovine papillomavirus type 1 (BPV-1). However, we demonstrate that the analogous residues required for E1 dimerization in BPV-1 and the low risk HPV-11 are also required for HPV-18 E1. We also present evidence that the HPV-18 E1 DNA-binding domain does not share the same nucleotide and amino acid requirements for specific DNA recognition as BPV-1 and HPV-11 E1.

Base Sequence↗

PubMed1995

The DpsA protein of Synechococcus sp. Strain PCC7942 is a DNA-binding hemoprotein. Linkage of the Dps and bacterioferritin protein families.

The Dps family of proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long term nutrient limitation. Some members of the Dps protein family have been shown to form large (approximately 150-kDa), hexameric complexes that bind chromosomal DNA with little sequence specificity. In this paper we report the nucleotide sequence of the dpsA gene from Synechococcus sp. PCC7942 encoding a cyanobacterial Dps homolog. The deduced amino acid sequence of the Synechococcus sp. DpsA protein revealed that a carboxyl-terminal domain of the protein was > 60% homologous to the COOH-terminal half of bacterioferritin. Other known Dps family members lack such high similarity to the bacterioferritins. Purification and spectroscopic analysis of the Synechococcus sp. DpsA protein complex revealed that the complex contains heme and has a weak catalase activity in vitro. Activity staining of nondenaturing polyacrylamide gels showed that the protein complex comigrated with both the heme and the catalase activity, and O2 evolution measurements yielded a maximal specific activity of 1.7 mumol of H2O2 consumed/micrograms of protein-1 min-1. We speculate that the protein may have a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggest that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Last, the evolutionary link between the Dps protein family and the bacterioferritins is discussed.

Amino Acid Sequence↗

PubMed1999

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex. The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.

Animals↗

PubMed1993

Increased sequence-specific p53-DNA binding activity after DNA damage is attenuated by phorbol esters.

Damage to cellular DNA greatly increases the levels of the tumor-suppressor gene p53 and induces cell cycle arrest in G1. A critical function of wild-type p53 is its ability to bind to specific DNA sequences. The effect of DNA damage on the sequence-specific DNA-binding properties of cellular p53 was investigated using DNA gel mobility-shift assays with nuclear extracts from NIH-3T3 cells. DNA damage (initiated by radiation) induced a rapid, cycloheximide-sensitive increase in the levels of nuclear p53-DNA binding activity and an increase in the half-life of the p53 protein. Increased p53-DNA binding activity could be detected at low (0.2 Gy), non-lethal doses of radiation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) attenuated the DNA damage-induced increase in p53-DNA binding activity by decreasing the half-life of the p53 protein. The tumor promoter properties of TPA may therefore be mediated by interfering with the cellular p53 response to DNA damage. The increased levels of p53 bound to specific DNA sequences following DNA damage may induce cell cycle arrest. p53-mediated growth arrest could occur by inhibition of DNA replication and/or alterations in transcription of cell cycle genes.

3T3 Cells↗

PubMed1986

DNA binding by the bacteriophage SPO1-encoded type II DNA-binding protein, transcription factor 1. Site-specific binding requires 5-hydroxymethyluracil-containing DNA.

The bacteriophage SPO1-encoded Type II DNA-binding protein, transcription factor 1 (TF1), forms complexes with specific sites in SPO1 DNA. We have investigated the binding of TF1 to one of its preferred sites in which the normal 5-hydroxymethyluracil (hmUra) of SPO1 DNA has been replaced by thymine and have also investigated the binding of a bacterial Type II DNA-binding protein (from Bacillus stearothermophilus) to the hmUra- and thymine-containing forms of the same DNA segment. Our results show that TF1 binds selectively to this high affinity binding site only in hmUra-containing DNA and that the bacterial Type II DNA-binding protein interacts nonspecifically with both forms of DNA.

Binding Sites↗

PubMed1996

Redox regulation of GA-binding protein-alpha DNA binding activity.

We have investigated the reduction/oxidation (redox) regulation of the heteromeric transcription factor GA-binding protein (GABP). GABP, also known as nuclear respiratory factor 2, regulates the expression of nuclear encoded mitochondrial proteins involved in oxidative phosphorylation, including cytochrome c oxidase subunits IV and Vb, as well as the expression of mitochondrial transcription factor 1. GABP is composed of two subunits, the Ets-related GABP-alpha, which mediates specific DNA binding, and GABP-beta, which forms heterodimers and heterotetramers on DNA sequences containing the PEA3/Ets motif ((C/A)GGA(A/T)(G/A)). We demonstrate here that GABP DNA binding activity and GABP-dependent gene expression in 3T3 cells are inhibited by pro-oxidant conditions. DNA binding of recombinant GABP-alpha was activated by chemical reduction (dithiothreitol) and by thioredoxin; however, GSSG inhibited GABP DNA binding activity. Treatment of GABP-alpha, but not GABP-beta1, with sulfhydryl-alkylating agents also inhibited GABP DNA binding activity. Our results suggest that GABP DNA binding activity is redox-regulated in vivo, possibly by thioredoxin-mediated reduction and by GSSG-mediated oxidation of the GABP-alpha subunit. The regulation of GABP (nuclear respiratory factor 2) DNA binding activity by cellular redox changes provides an important link between mitochondrial and nuclear gene expression and the redox state of the cell.

3T3 Cells↗

PubMed1994

A group of chromosomal proteins is specifically released by spermine and loses DNA-binding activity upon phosphorylation.

Biologically relevant concentrations as low as 500 microM spermine led to the specific release of chromatin-associated proteins from nuclei of rice (Oryza sativa) seedlings. Using a southwestern technique, it was shown that several of these proteins bind DNA. This affinity was lost upon in organello phosphorylation by an endogenous kinase. The effect of spermine was very specific. Spermidine was far less effective and putrescine was essentially ineffective in releasing these proteins. The most abundant spermine-released protein was shown to be homologous to the maize HMG1 protein. Our results suggest that spermine induces the release of spermine-released proteins by changing DNA conformation. Binding of these proteins might be sensitive to long-range changes in chromosome structure caused by torsional stress.

Adenosine Triphosphate↗

PubMed1990

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

Bacillus subtilis↗