Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q02556 (DNA-binding domain)
 6,431 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.
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PMID:Mapping the transcriptional transactivation function of simian virus 40 large T antigen. 185 53

The biochemical activities of a series of transformation-competent, replication-defective large T-antigen point mutants were examined. The assays employed reflect partial reactions required for the in vitro replication of simian virus 40 (SV40) DNA. Mutants which failed to bind specifically to SV40 origin sequences bound efficiently to single-stranded DNA and exhibited nearly wild-type levels of helicase activity. A mutation at proline 522, however, markedly reduced ATPase, helicase, and origin-specific unwinding activities. This mutant bound specifically to the SV40 origin of replication, but under certain conditions it was defective in binding to both single-stranded DNA and the partial duplex helicase substrate. This suggests that additional determinants outside the amino-terminal-specific DNA-binding domain may be involved in nonspecific binding of T antigen to single-stranded DNA and demonstrates that origin-specific DNA binding can be separated from binding to single-stranded DNA. A mutant containing a lesion at residue 224 retained nearly wild-type levels of helicase activity and recognized SV40 origin sequences, yet it failed to function in an origin-specific unwinding assay. This provides evidence that origin recognition and helicase activities are not sufficient for unwinding to occur. The distribution of mutant phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions provided by large T antigen.
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PMID:Large T-antigen mutants define multiple steps in the initiation of simian virus 40 DNA replication. 255 Jun 64

We have examined the role of the human papovavirus BK virus (BKV) tumor (T) antigen(s) in the maintenance of transformation and have identified the domain of T antigen essential for transformation. BKV-transformed BHK 21 and NIH 3T3 cells expressing antisense T-antigen RNA lose their ability to grow in soft agar, indicating the need for the continued expression of T antigen for the maintenance of the transformed phenotype. Experiments using translation termination linker insertion and deletion mutagenesis of BKV T antigen demonstrate that amino acids 356 to 384 are essential for transformation. Although BKV T antigen shares 100, 95, and 82% amino acid homology with that of simian virus 40 (SV40) for the nuclear localization signal, p53-binding domain, and DNA-binding domain, respectively, the transformation domains of BKV and SV40 T antigens share only 54% homology. Also, BKV T antigen lacks a substantial portion of the ATPase domain of SV40, and our results indicate the dispensability of the remaining portion for transformation by this protein. We suggest that the differences in the amino acids in the identified transformation domains together with the differences in the ATPase domains may account for the differences in the transformation potentials of the two proteins.
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PMID:Functional role of BK virus tumor antigens in transformation. 284 74

Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer. In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms. Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.
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PMID:Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain. 760 83

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.
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PMID:Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter. 787 28

DNA polymerases alpha, delta and epsilon from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physiochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (Km values) and specific inhibitors (Ki values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases alpha and epsilon from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells, when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lower Ki values, indicating lower affinity to deoxyribonucleoside 5'-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K50 values). Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases delta and epsilon was measured using poly(dA.dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.
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PMID:Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells by inhibitors of cell proliferation. 857 84

Previously, we reported on the discovery and characterization of a mammalian chromatin-associated protein, CHD1 (chromo-ATPase/helicase-DNA-binding domain), with features that led us to suspect that it might have an important role in the modification of chromatin structure. We now report on the characterization of the Drosophila melanogaster CHD1 homologue (dCHD1) and its localization on polytene chromosomes. A set of overlapping cDNAs encodes an 1883-aa open reading frame that is 50% identical and 68% similar to the mouse CHD1 sequence, including conservation of the three signature domains for which the protein was named. When the chromo and ATPase/helicase domain sequences in various CHD1 homologues were compared with the corresponding sequences in other proteins, certain distinctive features of the CHD1 chromo and ATPase/helicase domains were revealed. The dCHD1 gene was mapped to position 23C-24A on chromosome 2L. Western blot analyses with antibodies raised against a dCHD1 fusion protein specifically recognized an approximately 210-kDa protein in nuclear extracts from Drosophila embryos and cultured cells. Most interestingly, these antibodies revealed that dCHD1 localizes to sites of extended chromatin (interbands) and regions associated with high transcriptional activity (puffs) on polytene chromosomes from salivary glands of third instar larvae. These observations strongly support the idea that CHD1 functions to alter chromatin structure in a way that facilitates gene expression.
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PMID:CHD1 is concentrated in interbands and puffed regions of Drosophila polytene chromosomes. 869 58

The SNF2/SWI2 ATPase/helicase family comprises proteins from a variety of species, which serve a number of functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Several proteins with unknown functions are also included in this family. The number of genes that belong to this family is rapidly expanding, which makes it easier to analyze the common biological functions of the family members. This study was designed to clone the SNF2/SWI2 helicase-related genes from the fission yeast Schizosaccharomyces pombe in the hope that this would help to elucidate the common functions of the proteins in this family. The hrp1+ (helicase-related gene from S. pombe) gene was initially cloned by PCR amplification using degenerate primers based on conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. The hrp1+ ORF codes for an 1373-amino acid polypeptide with a molecular mass of 159 kDa. Like other SNF2/SWI2 family proteins, the deduced amino acid sequence of Hrp1 contains DNA-dependent ATPase/7 helicase domains, as well as a chromodomain and a DNA-binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-binding protein 1), suggesting that Hrp1 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. Northern blot analysis showed that the hrp1+ gene produces a 4.6-kb transcript, which reaches its maximal level just before the cells enter the exponential growth phase, and then decreases gradually. DNA-damaging agents, such as MMS, MNNG and UV, decrease the rate of transcription of hrp1+. Deletion of the hrp1+ gene resulted in accelerated cell growth. On the other hand, overexpression of Hrp1 caused a reduction in growth rate. These results indicate that hrp1+ may act as a negative regulator of cellular growth.
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PMID:Isolation and characterization of hrp1+, a new member of the SNF2/SWI2 gene family from the fission yeast Schizosaccharomyces pombe. 952 Feb 66

The yeast ENA1/PMR2A gene encodes a cation extrusion ATPase in Saccharomyces cerevisiae which is essential for survival under salt stress conditions. One important mechanism of ENA1 transcriptional regulation is based on repression under normal growth conditions, which is relieved by either osmotic induction or glucose starvation. Analysis of the ENA1 promoter revealed a Mig1p-binding motif (-533 to -544) which was characterized as an upstream repressing sequence (URSMIG-ENA1) regulated by carbon source. Its function was abolished in a mig1 mig2 double-deletion strain as well as in either ssn6 or tup1 single mutants. A second URS at -502 to -513 is responsible for transcriptional repression regulated by osmotic stress and is similar to mammalian cyclic AMP response elements (CREs) that are recognized by CREB proteins. This URSCRE-ENA1 element requires for its repression function the yeast CREB homolog Sko1p (Acr1p) as well as the integrity of the Ssn6p-Tup1p corepressor complex. When targeted to the GAL1 promoter by fusing with the Gal4p DNA-binding domain, Sko1p acts as an Ssn6/Tup1p-dependent repressor regulated by osmotic stress. A glutathione S-transferase-Sko1 fusion protein binds specifically to the URSCRE-ENA1 element. Furthermore, a hog1 mitogen-activated protein kinase deletion strain could not counteract repression on URSCRE-ENA1 during osmotic shock. The loss of SKO1 completely restored ENA1 expression in a hog1 mutant and partially suppressed the osmotic stress sensitivity, qualifying Sko1p as a downstream effector of the HOG pathway. Our results indicate that different signalling pathways (HOG osmotic pathway and glucose repression pathway) use distinct promoter elements of ENA1 (URSCRE-ENA1 and URSMIG-ENA1) via specific transcriptional repressors (Sko1p and Mig1/2p) and via the general Ssn6p-Tup1p complex. The physiological importance of the relief from repression during salt stress was also demonstrated by the increased tolerance of sko1 or ssn6 mutants to Na+ or Li+ stress.
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PMID:Repressors and upstream repressing sequences of the stress-regulated ENA1 gene in Saccharomyces cerevisiae: bZIP protein Sko1p confers HOG-dependent osmotic regulation. 985 77

The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation. We report here that brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter. Regulation of Antennapedia by BRM and OSA proteins requires sequences 5' to the P2 promoter. Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation. The OSA protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila DRI proteins. We propose that the OSA protein may target the BRM complex to Antennapedia and other regulated genes.
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PMID:The trithorax group gene osa encodes an ARID-domain protein that genetically interacts with the brahma chromatin-remodeling factor to regulate transcription. 989 21


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