scholarly journals Sequence Requirements for Interaction of Human Herpesvirus 7 Origin Binding Protein with the Origin of Lytic Replication

2001 ◽  
Vol 75 (8) ◽  
pp. 3925-3936 ◽  
Author(s):  
Laurie T. Krug ◽  
Naoki Inoue ◽  
Philip E. Pellett
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Human Herpesvirus ◽  
Binding Activity ◽  
Lytic Replication ◽  
Heteroduplex Analysis ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Origin Binding Protein

ABSTRACT As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBPH7) with its cognate sites in the 600-bp HHV-7oriLyt. We expressed the carboxyl-terminal domain of OBPH7 and found that amino acids 484 to 787 of OBPH7 were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBPH7 has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBPH6B), which binds with similar affinity to its two cognate OBP sites in the HHV-6BoriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBPH7 consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBPH6B consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBPH7interacts along one face of the DNA helix, with the major groove, as do OBPH6B and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBPH7 and OBPH6B.

scholarly journals Drosophila Mi-2 Negatively Regulates dDREF by Inhibiting Its DNA-Binding Activity

2002 ◽  
Vol 22 (14) ◽  
pp. 5182-5193 ◽  
Author(s):  
Fumiko Hirose ◽  
Nobuko Ohshima ◽  
Eun-Jeong Kwon ◽  
Hideki Yoshida ◽  
Masamitsu Yamaguchi
Keyword(s):  
Dna Binding ◽  
Polytene Chromosomes ◽  
Ectopic Expression ◽  
Binding Activity ◽  
Mobility Shift ◽  
Interacting Protein ◽  
Reciprocal Regulation ◽  
Dna Binding Activity ◽  
Expression Of Genes ◽  
Biochemical Analyses

ABSTRACT Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5′-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation

2001 ◽  
Vol 29 (6) ◽  
pp. 688-691 ◽  
Author(s):  
K. J. Campbell ◽  
N. R. Chapman ◽  
N. D. Perkins
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Uv Light ◽  
Binding Protein ◽  
Nuclear Factor Κb ◽  
Camp Response Element Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Dna Binding Activity

The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.

scholarly journals A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1

1994 ◽  
Vol 14 (3) ◽  
pp. 1852-1860
Author(s):  
K Nakagomi ◽  
Y Kohwi ◽  
L A Dickinson ◽  
T Kohwi-Shigematsu
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Contact ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Sequence Context ◽  
Dna Binding Activity

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

scholarly journals Redox Regulation of GA-binding Protein-α DNA Binding Activity

1996 ◽  
Vol 271 (41) ◽  
pp. 25617-25623 ◽  
Author(s):  
Mark E. Martin ◽  
Yurii Chinenov ◽  
Mi Yu ◽  
Tonya K. Schmidt ◽  
Xiu-Ying Yang
Keyword(s):  
Dna Binding ◽  
Redox Regulation ◽  
Binding Protein ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Ga Binding Protein

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells

1992 ◽  
Vol 12 (9) ◽  
pp. 4104-4111
Author(s):  
L Sistonen ◽  
K D Sarge ◽  
B Phillips ◽  
K Abravaya ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Hsp70 Gene ◽  
Mobility Shift ◽  
Heat Shock Element ◽  
Dna Binding Activity ◽  
Erythroleukemia Cells

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

scholarly journals DNA Binding Activity of the Herpes Simplex Virus Type 1 Origin Binding Protein, UL9, Can Be Modulated by Sequences in the N Terminus: Correlation between Transdominance and DNA Binding

2006 ◽  
Vol 80 (9) ◽  
pp. 4491-4500 ◽  
Author(s):  
Soma Chattopadhyay ◽  
Sandra K. Weller
Keyword(s):  
Herpes Simplex ◽  
Dna Binding ◽  
Binding Activity ◽  
Plaque Formation ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Origin Binding Protein ◽  
N Terminus ◽  
Simplex Virus

ABSTRACT UL9, the origin binding protein of herpes simplex virus type 1, is a member of the SF2 family of helicases. Cotransfection of cells with infectious viral DNA and plasmids expressing either full-length UL9 or the C-terminal DNA binding domain alone results in the drastic inhibition of plaque formation which can be partially relieved by an insertion mutant lacking DNA binding activity. In this work, C-terminally truncated mutants which terminate at or near residue 359 were shown to potentiate plaque formation, while other C-terminal truncations were inhibitory. Thus, residues in the N-terminal region appear to regulate the inhibitory properties of UL9. To identify which residues were involved in this regulation, a series of N-terminally truncated mutants were constructed which contain the DNA binding domain and various N-terminal extensions. Mutants whose N terminus is either at residue 494 or 535 were able to bind the origin efficiently and were inhibitory to plaque formation, whereas constructs whose N terminus is at residue 304 or 394 were defective in origin binding activity and were able to relieve inhibition. Since UL9 is required for viral infection at early but not late times and is inhibitory to infection when overexpressed, we propose that the DNA binding activities of UL9 are regulated during infection. For infection to proceed, UL9 may need to switch from a DNA binding to a non-DNA binding mode, and we suggest that sequences residing in the N terminus play a role in this switch.

scholarly journals DNA-binding activity in the excretory–secretory products ofTrichinella pseudospiralis(Nematoda: Trichinelloidea)

Parasitology ◽  
2001 ◽  
Vol 123 (3) ◽  
pp. 301-308 ◽  
Author(s):  
C. H. MAK ◽  
R. C. KO
Keyword(s):  
Dna Binding ◽  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Binding Activity ◽  
Mobility Shift ◽  
Competition Analysis ◽  
Dna Binding Activity ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Secretory Products

A novel DNA-binding peptide ofMr∼30 kDa was documented for the first time in the excretory–secretory (E–S) products of the infective-stage larvae ofTrichinella pseudospiralis.Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E–S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of theMrof proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E–S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E–S product.

scholarly journals Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors

2000 ◽  
Vol 20 (11) ◽  
pp. 3870-3879 ◽  
Author(s):  
Sheng-Fu Wang ◽  
Stephen Ayer ◽  
William A. Segraves ◽  
Daryl R. Williams ◽  
Alexander S. Raikhel
Keyword(s):  
Dna Binding ◽  
Nuclear Receptors ◽  
Hormone Receptors ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Receptor Complex ◽  
S2 Cells ◽  
Ligand Specificity ◽  
Mobility Shift ◽  
Dna Binding Activity

ABSTRACT The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than theDrosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series ofAedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in AedesEcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.

scholarly journals The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication

2004 ◽  
Vol 85 (7) ◽  
pp. 2001-2013 ◽  
Author(s):  
Koen W. R. van Cleef ◽  
Wendy M. A. Scaf ◽  
Karen Maes ◽  
Suzanne J. F. Kaptein ◽  
Erik Beuken ◽  
...  
Keyword(s):  
Dna Binding ◽  
Virus Replication ◽  
Deletion Mutant ◽  
Nuclear Protein ◽  
Human Herpesvirus ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Double Stranded Dna

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

scholarly journals The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

1994 ◽  
Vol 14 (7) ◽  
pp. 4380-4389 ◽  
Author(s):  
L I Chen ◽  
T Nishinaka ◽  
K Kwan ◽  
I Kitabayashi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Product ◽  
Binding Activity ◽  
Negative Regulator ◽  
Control Element ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Mobility Shift Assay

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

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