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Results for “DNA binding”

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PubMed1995

The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1.

The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.

Adenovirus E2 Proteins↗

PubMed1995

Xenopus interspersed RNA families, Ocr and XR, bind DNA-binding proteins.

Interspersed RNA makes up two-thirds of cytoplasmic polyadenylated RNA in Xenopus and sea urchin eggs. Although it has no known function, previous work has suggested that at least one family of interspersed RNA, XR, binds Xenopus oocyte proteins, and can influence the rate of translation. We have used two Xenopus repeat families, Ocr and XR, to explore their protein binding abilities. Ocr RNA binds the same pattern of highly abundant oocyte proteins that XR RNA binds, which are believed to be messenger ribonucleoprotein (mRNP) particle proteins. In addition, we show that Ocr RNA binds the Oct-60 protein, a member of the POU-domain family of transcription factors found in Xenopus oocytes. Using a 32 base pair sequence from the XR repeat in a DNA affinity column two proteins were isolated, 66 kDa and 92 kDa, that together form a complex with XR DNA. One of these proteins (92 kDa) also binds XR RNA. We suggest that the role of at least a subset of interspersed RNAs in development may be to bind, and sequester in the cytoplasm, DNA-binding proteins until the end of oogenesis.

Animals↗

PubMed2003

Yeast xrs2 binds DNA and helps target rad50 and mre11 to DNA ends.

Saccharomyces cerevisiae Rad50, Mre11, and Xrs2 proteins are involved in homologous recombination, non-homologous end-joining, DNA damage checkpoint signaling, and telomere maintenance. These proteins form a stable complex that has nuclease, DNA binding, and DNA end recognition activities. Of the components of the Rad50.Mre11.Xrs2 complex, Xrs2 is the least characterized. The available evidence is consistent with the idea that Xrs2 recruits other protein factors in reactions that pertain to the biological functions of the Rad50.Mre11.Xrs2 complex. Here we present biochemical evidence that Xrs2 has an associated DNA-binding activity that is specific for DNA structures. We also define the contributions of Xrs2 to the activities of the Rad50.Mre11.Xrs2 complex. Importantly, we demonstrate that Xrs2 is critical for targeting of Rad50 and Mre11 to DNA ends. Thus, Xrs2 likely plays a direct role in the engagement of DNA substrates by the Rad50. Mre11.Xrs2 complex in various biological processes.

Adenosine Triphosphatases↗

PubMed1993

Repression of major histocompatibility complex IA expression by glucocorticoids: the glucocorticoid receptor inhibits the DNA binding of the X box DNA binding protein.

Glucocorticoids are effective repressors of major histocompatibility complex (MHC) class II gene expression. The repression occurs in B cells, which constitutively express MHC class II, as well as in macrophages, which only express MHC class II after the cells are treated with interferon gamma. For the MHC class II gene IA beta, this negative regulation has been linked to the X box DNA sequence, located with the IA beta promoter. The addition of the glucocorticoid receptor was shown to inhibit the DNA binding of the X box DNA binding protein to the X box. The DNA binding of two other DNA binding proteins that recognize elements within this promoter was unaffected by the addition of glucocorticoid receptor. It is likely that the repression of IA beta gene expression by glucocorticoids occurs because the X box DNA binding protein is prevented from binding to the DNA and activating transcription.

Animals↗

PubMed1984

Subcellular distribution of DNA-binding and non-DNA-binding 1,25-dihydroxyvitamin D receptors in chicken intestine.

A comparison of 1,25-dihydroxyvitamin D3 receptor concentration and equilibrium dissociation constants of whole tissue, nuclear, and cytosol extracts of vitamin D-deficient chicken intestine has been carried out. The extracts had the following order of receptor concentration: nuclei greater than whole tissue greater than cytosol. The receptors in each preparation had identical equilibrium dissociation constants (Kd) for 1,25-dihydroxyvitamin D3. However, the receptor in the cytosol fraction did not bind to DNA-cellulose, while a large fraction of the receptor population in crude nuclear extract and whole tissue extract bound to DNA-cellulose and could be eluted with 0.22 M KCl, suggesting that the cytosolic form of the receptor does not possess a DNA binding site.

Animals↗

PubMed1994

Relationship between zinc content and DNA-binding activity of the DNA-binding motif of the transcription factor ALCR in Aspergillus nidulans.

The transcription factor ALCR of the ethanol utilisation pathway in Aspergillus nidulans contains a zinc binuclear motif (CysX2CysX6CysX16CysX2CysX6Cys), within the DNA-binding domain located in the N-terminal region of the ALCR protein. Specific targets have been localised in the promoter of the alcR gene, involved in the autoregulation process, and in the promoter of the structural gene alcA (encoding alcohol dehydrogenase I), which is also under the control of ALCR. The DNA-binding domain has been expressed in-Escherichia coli as a GST-ALCR (7-58*) fusion protein and also obtained as an ALCR (7-58*) peptide. Both the ALCR fusion protein and the ALCR peptide are able to bind 65Zn(II) in vitro, if reduction of cysteines occurs prior to the addition of zinc. Competition experiments showed that Cd(II), Co(II) and Cu(II) are efficient competitors for the zinc binding sites. The ALCR DNA-binding domain was shown to contain 2 mol of tightly bound Zn(II) per mole of fusion protein. Removal of the intrinsic Zn(II) requires treatment with Chelex. This treatment abolishes the ability of the protein to bind to the targets of ALCR located in the alcA and alcR promoters. The apo-ALCR DNA-binding motif could be reconstituted with Zn(II) or Cd(II), restoring specific DNA binding to both types of targets. Thus a direct relationship was shown to exist between the zinc content of ALCR and its DNA-binding activity.

Alcohol Dehydrogenase↗

PubMed1998

Characterization of a novel DNA binding domain within the amino-terminal region of the RAG-1 protein.

Rag-1 and Rag-2 are the critical components of the V-(D)-J recombinase required for site-specific recombination of the antigen receptor genes. In this study, we have examined the ability of recombinant (r) Rag-1 and Rag-2 to bind the recombination signal sequences (RSS) and have determined that rRag-1, but not rRag-2, is able to directly bind DNA. rRAG-1 DNA binding activity was found to reside within a novel amino-terminal arginine-rich (RR) domain with partial homology to a variety of nucleic acid binding domains. Although the RR-domain did not demonstrate RSS-specificity, this DNA binding domain may stabilize the interaction of RAG-1 with, or increase the affinity for, the V-(D)-J recombination signals.

Amino Acid Sequence↗

PubMed2002

A common DNA-binding site for SZF1 and the BRCA1-associated zinc finger protein, ZBRK1.

More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome. KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins. However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs. Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1. Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response. The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site. The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity. Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences. Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo.

3T3 Cells↗

PubMed1993

Identification of a DNA binding domain in simian virus 40 capsid proteins Vp2 and Vp3.

We have identified both biochemically and genetically a protein domain within the simian virus 40 virion protein Vp3, and within Vp2 since its carboxyl two-thirds are identical to the full-length Vp3, that binds DNA in a sequence nonspecific manner. Both the Vp2 and Vp3 (Vp2/3) components of SV40 and mutant SV40(202T) bound either SV40 or pBR322 DNA equally well. Wild type and mutant Vp2/3 proteins, expressed as fusion proteins with glutathione S-transferase (GST), were tested for their ability to bind DNA. GST-Vp3 bound DNA at physiological salt concentrations with an apparent Kd of 2.5 x 10(-8) M and also bound RNA with 4-fold higher affinity. Over 90% of the nucleic acid binding, and all of the activity, was lost upon removal of the carboxyl-terminal 13 and 35 residues, respectively. The DNA binding domain was shown to be distinct and separable from the Vp2/3 nuclear transport signal since mutations within the nuclear transport signal that reduce or abolish nuclear localization of Vp2/3 had no effect on the DNA binding activity of mutant Vp2/3 fusion proteins. The carboxyl-terminal 40 residues of Vp2/3 in the form of a beta-galactosidase fusion protein, F6, are sufficient for DNA binding and may cause compaction of the DNA. The significance of this DNA binding and possible compaction are discussed in relation to the assembly of virion particles.

Animals↗

PubMed1993

Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

Amino Acid Sequence↗

PubMed2005

Inhibition of DNA binding by human estrogen-related receptor 2 and estrogen receptor alpha with minor groove binding polyamides.

Human estrogen-related receptor 2 (hERR2, ESRRB, ERRbeta, NR3B2) belongs to a class of nuclear receptors that bind DNA through sequence-specific interactions with a 5'-AGGTCA-3' estrogen response element (ERE) half-site in the major groove and an upstream 5'-TNA-3' site in the minor groove. This minor groove interaction is mediated by a C-terminal extension (CTE) of the DNA binding domain and is unique to the estrogen-related receptors. We have used synthetic pyrrole-imidazole polyamides, which bind specific sequences in the minor groove, to demonstrate that DNA binding by hERR2 is sensitive to the presence of polyamides in both the upstream minor groove CTE site and the minor groove of the ERE half-site. Thus, polyamides can inhibit hERR2 by two mechanisms, by direct steric blockage of minor groove DNA contacts mediated by the CTE and by changing the helical geometry of DNA such that major groove interactions are weakened. To confirm the generality of the latter approach, we show that the dimeric human estrogen receptor alpha (hERalpha, ESR1, NR3A1), which binds in the major groove of the ERE, can be inhibited by a polyamide bound in the opposing minor groove of the ERE. These results highlight two mechanisms for inhibition of protein-DNA interactions and extend the repertoire of DNA recognition motifs that can be inhibited by polyamides. These molecules may thus be useful for controlling expression of hERR2- or hERalpha-responsive genes.

Binding, Competitive↗

PubMed1995

Characterization and localization of the equine herpesvirus 1 major DNA binding protein.

In previous studies of equine herpesvirus 1 (EHV-1) gene regulation, we observed an abundant early infected cell polypeptide (ICP), designated ICP130, which appeared in reduced amounts in cells infected with defective interfering particle-rich EHV-1 stocks compared to standard EHV-1-infected cells. To characterize this ICP further, a monoclonal antibody (MAb) was developed to EHV-1 ICP130 and used to (1) affinity purify ICP130, (2) examine ICP130's ability to bind DNA, and (3) define the synthesis and intracellular localization of ICP130 during productive EHV-1 infections. Although anti-ICP130 MAbs did not crossreact with any HSV-1 protein in immunoblots, a polyclonal antiserum against HSV-2 major DNA binding protein (ICSP11,12) did react with purified EHV-1 ICP130. DNA band shift assays indicated that (1) the mobility of shifted bands representing DNA/EHV-1-infected cell protein complexes was further decreased by the addition of either anti-ICP130 MAbs or anti-ICSP11,12, but not by the addition of irrelevant MAbs, (2) the ability of ICP130 to complex with DNA was not sequence dependent, (3) ICP130 associated with both single- and double-stranded oligomers, and (4) similar supershifted patterns were produced using affinity-purified ICP130 and anti-ICP130 MAbs. During productive infection, ICP130 initially localized rapidly and exclusively to the infected cell's nucleus in a generalized, fine granular pattern. Over the course of infection, this pattern typically progressed to include several large, intensely reactive intranuclear granules, and by 6 hr p.i. some cytoplasmic reactivity also was visible. In < 5% of the cells, a dense, fibrillar network surrounding the nucleus was observed instead. The progressive changes in nuclear localization depended upon the onset of viral DNA replication, and once the late pattern was established, ongoing DNA synthesis was required to maintain it. The results indicate that ICP130 is the previously reported EHV-1 counterpart of the HSV major DNA-binding protein and is similar, but not identical, in many aspects.

Animals↗

PubMed1992

Members of the USF family of helix-loop-helix proteins bind DNA as homo- as well as heterodimers.

We have isolated human cDNA clones for USF2, a new member of the upstream stimulatory factor (USF) family of transcription factors. Analysis of these clones revealed the existence of highly conserved elements in the C terminal region of all USF proteins. These include the basic region, helix-loop-helix (HLH) motif, and, in the case of the human proteins, the C-terminal leucine repeat (LR). In addition, a highly conserved USF-specific domain is located immediately upstream of the basic region. Using in vitro translated proteins, we found that all members of the USF family bound DNA as dimers. The N-terminal portion of USF, including the USF-specific domain, was entirely dispensable for dimer formation and DNA-binding. However, deletion mutants of USF2 lacking the LR were deficient in DNA-binding activity. Interestingly, each of the USF proteins could form functional heterodimers with the other family members, including the sea urchin USF, which does not have a LR motif. This indicates that the conserved LR in human USF is not required for dimer formation, and influences only indirectly DNA-binding.

Amino Acid Sequence↗

PubMed1992

Chelatable metal ions are not required for aryl hydrocarbon receptor transformation to a DNA binding form: phenanthrolines are possible competitive antagonists of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds by binding DNA and altering gene transcription. We determined whether AhR transformation to a DNA binding form requires chelatable metal ions. The chelator 1,10-phenanthroline and its nonchelating isomers 1,7- and 4,7-phenanthroline blocked, in a concentration-dependent manner, TCDD-elicited transformation of the AhR in rat hepatic cytosol to a form which bound a dioxin-response element (DRE; upstream of the structural gene for cytochrome P4501A1). This was found to be due to the ability of these compounds to competitively inhibit [3H]TCDD specific binding to the AhR under conditions in vitro. EDTA (20 mM) failed to inhibit DRE binding of the transformed AhR, but pretreatment of cytosol with EDTA prior to transformation inhibited DRE binding up to 60%. However, removal of EDTA from the cytosol by gel filtration prior to incubation with TCDD resulted in the same DRE binding as filtered control cytosol without the added divalent metal ions. Both chelators, oxalic acid and iminodiacetic acid, failed to inhibit DRE binding when added prior to AhR transformation. Together these data indicate that chelatable metal ions are not required for AhR transformation to the DNA binding form.

Animals↗

PubMed1991

Dominant negative mutations in yeast TFIID define a bipartite DNA-binding region.

Genetic analysis showed that the conserved C-terminal 180 amino acids of yeast TFIID contain all the essential functions for growth of yeast and response to acidic transcriptional activation signals. A genetic screen was used to identify functionally important residues within this C-terminal region. Five dominant TFIID mutations were isolated that had lost the ability to bind DNA. Four of these mutations were single amino acid substitutions in the most N-terminal of two 66-67 amino acid repeats in TFIID. Analogous mutations made in the most C-terminal repeat all failed to bind DNA and inhibited growth of cells, suggesting that the DNA-binding function of TFIID is partitioned between the two repeated regions. Overproduction of wild-type TFIID rescued the dominance of the TFIID mutants, suggesting that the mutant proteins are dominant because they compete with wild-type TFIID for binding to one or more essential transcription factors.

Amino Acid Sequence↗

PubMed1997

Quercetin inhibits heat shock protein induction but not heat shock factor DNA-binding in human breast carcinoma cells.

The flavonoid quercetin inhibits the heat-induced synthesis of heat shock proteins (hsps) in a variety of cell lines. To determine whether quercetin could inhibit hsp expression in breast cancer cells, we used the human breast cancer cell line, MDA-MB-231. Treatment of these cells with quercetin decreased the heat-induced synthesis of hsp27 and hsp70. However, inhibition of hsp expression did not correspond with the reduced ability of heat shock transcription factors (HSFs) to bind DNA. Furthermore, while quercetin treatment inhibited HSF2 expression, it only slightly affected HSF1 expression in breast cancer cells. In contrast, quercetin inhibited both HSF DNA-binding activity and HSF expression in HeLa cells. Our studies suggest that quercetin's action is cell-type specific, and in breast cancer cells may involve regulation of HSF transcriptional activity, rather than regulation of its DNA-binding activity.

Binding Sites↗

PubMed1983

Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor.

Amino acid and DNA sequence comparisons suggest that many sequence-specific DNA-binding proteins have in common an homologous region of about 22 amino acids. This region corresponds to two consecutive alpha-helices that occur in both Cro and cI repressor proteins of bacteriophage lambda and in catabolite gene activator protein of Escherichia coli and are presumed to interact with DNA. The results obtained here suggest that this alpha-helical DNA-binding fold occurs in many proteins that regulate gene expression. It also appears that this DNA-binding unit evolved from a common evolutionary precursor.

Amino Acid Sequence↗

PubMed1998

Transcriptional repression by v-Ski and c-Ski mediated by a specific DNA binding site.

The Ski oncoprotein has been shown to bind DNA and activate transcription in conjunction with other cellular factors. Because tumor cells or myogenic cells were used for those studies, it is not clear that those activities of Ski are related to its transforming ability. In this study, we use a nuclear extract of c-ski-transformed cells to identify a specific DNA binding site for Ski with the consensus sequence GTCTAGAC. We demonstrate that both c-Ski and v-Ski in nuclear extracts are components of complexes that bind specifically to this site. By evaluating the features of the sequence that are critical for binding, we show that binding is cooperative. Although Ski cannot bind to this sequence on its own, we use cross-linking with ultraviolet light to show that Ski binds to this site along with several unidentified cellular proteins. Furthermore, we find that Ski represses transcription either through upstream copies of this element or when brought to the promoter by a heterologous DNA binding domain. This is the first demonstration that Ski acts as a repressor rather than an activator and could provide new insights into regulation of gene expression by Ski.

Animals↗