The DNA-binding domain of the yeast Spt10p activator includes a zinc finger that is homologous to foamy virus integrase

The yeast SPT10 gene encodes a putative histone acetyltransferase that binds specifically to pairs of upstream activating sequence (UAS) elements found only in the histone gene promoters. Here, we demonstrate that the DNA-binding domain of Spt10p is located between residues 283 and 396 and includes a His(2)-Cys(2) zinc finger. The binding of Spt10p to the histone UAS is zinc-dependent and is disabled by a zinc finger mutation (C388S). The isolated DNA-binding domain binds to single histone UAS elements with high affinity. In contrast, full-length Spt10p binds with high affinity only to pairs of UAS elements with very strong positive cooperativity and is unable to bind to a single UAS element. This implies the presence of a "blocking" domain in full-length Spt10p, which forces it to search for a pair of UAS elements. Chromatin immunoprecipitation experiments indicate that, unlike wild-type Spt10p, the C388S protein does not bind to the promoter of the gene encoding histone H2A (HTA1) in vivo. The C388S mutant has a phenotype similar to that of the spt10Delta mutant: poor growth and global aberrations in gene expression. Thus, the C388S mutation disables the DNA-binding function of Spt10p in vitro and in vivo. The zinc finger of Spt10p is homologous to that of foamy virus integrase, perhaps suggesting that this integrase is also a sequence-specific DNA-binding protein.     

Electron paramagnetic resonance characterization of the copper-resistance protein PcoC from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Continuous-wave and pulsed electron paramagnetic resonance have been applied to the study of the Cu(II) site of the copper-resistance protein PcoC from Escherichia coli and certain variant forms. Electron spin echo envelope modulation (ESEEM) experiments confirm the presence of two histidine ligands, His1 and His92, at the Cu(II) site of wild-type PcoC, consistent with the available X-ray crystallographic data for the homolog CopC (67% sequence identity) from Pseudomonas syringae pv. tomato. The variants H1F and H92F each lack one of the histidine residues close to the Cu(II) site. The ESEEM data suggest that the surviving histidine residue remains as a ligand. The nA variant features an extra alanine residue at the N terminus, which demotes the His1 ligand to position 2. At least one of the two histidine residues is bound at the Cu(II) site in this form. Simulation of the (14)N superhyperfine structure in the continuous-wave spectra confirms the presence of at least three nitrogen-based ligands at the Cu(II) sites of the wild-type, H92F and nA forms, while the H1F variant has two nitrogen ligands. The spectra of wild-type form can be fitted adequately with a 3N or a 4N model. The former is consistent with the crystal structure of the CopC homolog, where His1 acts as a bidentate ligand. The latter raises the possibility of an additional unidentified nitrogen ligand. The markedly different spectra of the H1F and nA forms compared with the wild-type and H92F proteins further highlight the integral role of the N-terminal histidine residue in the high-affinity Cu(II) site of PcoC.     

Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein.

We tested whether the selection of target sites can be manipulated by fusing retroviral integrase with a sequence-specific DNA-binding protein. A hybrid protein that has the Escherichia coli LexA protein fused to the C terminus of the human immunodeficiency virus type 1 integrase was constructed. The fusion protein, IN1-288/LA, retained the catalytic activities in vitro of the wild-type human immunodeficiency virus type 1 integrase (WT IN). Using an in vitro integration assay that included multiple DNA fragment as the target DNA, we found that IN1-288/LA preferentially integrated viral DNA into the fragment containing a DNA sequence specifically bound by LexA protein. No bias was observed when the LexA-binding sequence was absent, when the fusion protein was replaced by WT IN, or when LexA protein was added in the reaction containing IN1-288/LA. A majority of the integration events mediated by IN1-288/LA occurred within 30 bp of DNA flanking the LexA-binding sequence. The specificity toward the LexA-binding sequence and the distribution and frequency of target site usage were unchanged when the integrase component of the fusion protein was replaced with a variant containing a truncation at the N or C terminus or both, suggesting that the domain involved in target site selection resides in the central core region of integrase. The integration bias observed with the integrase-LexA hybrid shows that one effective means of altering the selection of DNA sites for integration is by fusing integrase to a sequence-specific DNA-binding protein.     

Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper.

Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.     

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.     

Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI

HinP1I, a type II restriction endonuclease, recognizes and cleaves a palindromic tetranucleotide sequence (G↓CGC) in double-stranded DNA, producing 2 nt 5′ overhanging ends. Here, we report the structure of HinP1I crystallized as one protein monomer in the crystallographic asymmetric unit. HinP1I displays an elongated shape, with a conserved catalytic core domain containing an active-site motif of SDX₁₈QXK and a putative DNA-binding domain. Without significant sequence homology, HinP1I displays striking structural similarity to MspI, an endonuclease that cleaves a similar palindromic DNA sequence (C↓CGG) and binds to that sequence crystallographically as a monomer. Almost all the structural elements of MspI can be matched in HinP1I, including both the DNA recognition and catalytic elements. Examining the protein–protein interactions in the crystal lattice, HinP1I could be dimerized through two helices located on the opposite side of the protein to the active site, generating a molecule with two active sites and two DNA-binding surfaces opposite one another on the outer surfaces of the dimer. A possible functional link between this unusual dimerization mode and the tetrameric restriction enzymes is discussed.     

Identification of proteins involved in the regulation of yeast iso- 1-cytochrome C expression by oxygen.

On the basis of a gel electrophoresis retardation assay, protein(s) which interact specifically with the upstream activating site (UASc) of the yeast iso-1-cytochrome C (CYC1) gene were identified and separated by heparin ultrogel chromatography. DNase I protection experiments indicate that these factors protect a 23-bp sequence overlapping the UASc site previously defined. The specific binding activity is strongly reduced in extracts prepared from a wild-type strain grown anaerobically. It is absent in a mutant strain blocked in the biosynthesis of heme but it is restored upon the addition of the missing precursor, delta amino levulinic acid (dALA) to the growth medium. In contrast, the binding activity does not differ significantly in extracts form a wild-type strain grown in either glucose or glycerol as carbon source. These data strongly argue that the CYC1 UAS binding protein(s) that we have identified mediate the oxygen and heme control of cytochrome C biosynthesis.     

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro.

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

Factors that determine cell-specific gene expression in pancreatic endocrine tumor cells

Phenotypically distinct islet tumor cell lines may recapitulate certain of the developmental pathways of normal islet cell differentiation by expressing a combinatorial set of positively and negatively acting DNA-binding proteins to allow for the programmed expression of genes encoding polypeptide hormones. The structure of one of these DNA-binding proteins, a cyclic AMP-responsive protein (CREB) that binds specific DNA regulatory elements in the somatostatin gene, has been deduced from the sequence of a cloned cDNA. The CREB protein contains a DNA-binding domain separate from a cAMP-dependent protein kinase A activation domain. Further characterizations of the genes encoding the DNA-binding proteins should help to elucidate the cellular processes involved in islet cell differentiation and the genesis of tumors.     

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.