Evidence for nuclear and DNA binding forms of the receptor-glucocorticoid complex from hepatoma tissue culture cells.

Binding to DNA associated with cellulose has been used to investigate the receptor-glucocorticoid complex isolated from a line of rat hepatoma tissue culture cells. The amount of activated complex that bound to DNA was approximately half that which bound to nuclei. Additional results suggest the existence of two forms of the activated glucocorticoid receptor-steroid complex in about equal amounts: one form binds only to nuclei and the other binds to DNA and nuclei. The two forms also differ in their stability, with the DNA/nuclei binging form being relatively labile. The binding of either form to the appropriate acceptor is reduced by cytosol inhibitors by the same mechanism.     

The conformations of the manganese transport regulator of Bacillus subtilis in its metal-free state

The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 A. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR.     

A new mechanism for steroid unresponsiveness: loss of nuclear binding activity of a steroid hormone receptor

The glucocorticoid, dexamethasone, binds to the specific cytosol receptors of a steroid-resistant mouse lymphoma cell line with the same affinity as to the receptors of the steroid-responsive parental cells. In the sensitive cells, the receptor-steroid complex translocates to the nucleus, whereas in the resistant cells nuclear transfer is greatly diminished. “Activated” receptor-dexamethasone complex from sensitive cells binds to isolated nuclei from both sensitive and resistant cell types, whereas the complex from the resistant cells binds to neither nuclei. Furthermore, the activated complex from sensitive cells binds to isolated homologous and heterologous DNA, whereas the complex from the resistant cells displays greatly reduced binding activity, implying that DNA plays a significant role in nuclear binding. These results suggest that the normal glucocorticoid receptor has two active domains: one for steroid binding, and the other for interaction with nuclear acceptor sites. The resistant cells described in this paper contain a receptor apparently defective in the latter activity.     

Induced fit, folding, and recognition of the NF-kappaB-nuclear localization signals by IkappaBalpha and IkappaBbeta

Protein structure prediction codes based on the associative memory Hamiltonian were used to probe the binding modes between the nuclear localization signal (NLS) polypeptide of NF-kappaB and the inhibitors IkappaBalpha and IkappaBbeta. Experimentally, it is known that the NLS polypeptide is unstructured in the NF-kappaB complex with DNA but it forms an extended helical structure with the NLS (residues 301-304) between the two helices in the NF-kappaB/IkappaBalpha complex. The simulations included the NF-kappaB(p65) and (p50) NLS polypeptides and various mutants alone and in the presence of IkappaBalpha and IkappaBbeta. The simulations predict that the NLS polypeptide by itself binds tightly to IkappaBalpha and IkappaBbeta. In the NF-kappaB (p50/p65) heterodimer, the p50 NLS is predicted to remain free to bind to importin alpha. In the interaction with IkappaBalpha, both p65 NLSs are predicted to be bound. In IkappaBbeta, the NLS polypeptide binds to two binding sites, as seen in the crystal structure, with one site heavily favored for stable binding.     

Interaction of DNA fragmentation factor (DFF) with DNA reveals an unprecedented mechanism for nuclease inhibition and suggests that DFF can be activated in a DNA-bound state

DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) and its chaperone/inhibitor DFF45 (ICAD-L) that can be activated during apoptosis to induce DNA fragmentation. Here, we demonstrate that DFF directly binds to DNA in vitro without promoting DNA cleavage. DNA binding by DFF is mediated by the nuclease subunit, which can also form stable DNA complexes after release from DFF. Recombinant and reconstituted DFF is catalytically inactive yet proficient in DNA binding, demonstrating that the nuclease subunit in DFF is inhibited in DNA cleavage but not in DNA binding, revealing an unprecedented mode of nuclease inhibition. Activation of DFF in the presence of naked DNA or isolated nuclei stimulates DNA degradation by released DFF40 (CAD). In transfected HeLa cells transiently expressed DFF associates with chromatin, suggesting that DFF could be activated during apoptosis in a DNA-bound state.     

Enhanced recA protein binding to Z DNA represents a kinetic perturbation of a general duplex DNA binding pathway

recA protein binding to duplex DNA is enhanced when a B form DNA substrate is replaced with a left-handed Z form helix. This represents a kinetic rather than an equilibrium effect. Binding to Z DNA is much faster than binding to B DNA. In other respects, binding to the two DNA forms is quite similar. recA protein binds to B or Z DNA with a stoichiometry of 1 monomer/4 base pairs. The final protein filament exhibits a right-handed helical structure when either B or Z form DNAs are bound. There are only two evident differences: the kcat for ATP hydrolysis is reduced 3-4-fold when Z DNA is bound, and recA binding at equilibrium is less stable on Z DNA than on B DNA. At steady state, the binding favors B DNA in competition experiments. The results indicate that Z DNA binding by recA protein follows the same pathway as for recA binding to B DNA, but that the nucleation step is faster on the Z form helix.     

Evidence for the presence of two active forms of cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) in rat kidney. Specialized functions of two CTBPs in intracellular T3 translocation

Cytosolic NADPH-dependent 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) purified from rat kidney was further characterized in its T3 binding and its interaction with nuclei. Pretreatment of the CTBP with NADP induced dithiothreitol (DTT)-dependent T3 binding. The DTT-dependent T3 binding was increased by NADP in a concentration-dependent manner, and the maximal binding was obtained by 0.1 microM NADP. Higher concentrations of NADP (more than 0.1 microM), however, reduced T3 binding. NAD also induced DTT-dependent T3 binding, but was very low compared to that induced by NADP. NADPH and NADH did not produce DTT-dependent T3 binding. This NADP-activated, DTT-dependent T3 binding was characterized as follows: Ka for T3 binding was 1.8 x 10(9) M-1, and the maximal binding capacity was 15,000 pmol/mg of protein in the CTBP activated by 0.1 microM NADP. The molecular weight of the CTBP was 58,000 (4.7 S). A complex of [125I]T3 and CTBP (NADP.DTT.CTBP.[125I]T3), which was made from the CTBP pretreated with NADP and DTT, did not bind to DNA. However, the complex bound to the nuclei prepared from rat kidney. Treatment of the nuclei with 0.38 M KCl and with DNase I did not lead to loss of the binding activity for the complex. Treatment of nuclei with 0.5 M NaCl led to the loss of the activity for binding the complex. A complex of [125I]T3 and NADPH-activated CTBP did not bind these nuclear preparations. These results suggested that the active form of CTBP is present in two different forms: one is NADPH-activated, which plays a role as a reservoir for cytoplasmic T3, and the other is NADP-activated, which plays a role as a T3 carrier protein that transfers T3 from cytoplasm to nucleus.     

Evidence for distinct sulfhydryl groups associated with steroid- and DNA-binding domains of rat thymus glucocorticoid receptors

We have found that nonactivated and activated forms of the rat thymus glucocorticoid-receptor complex (GRC) will react with reactive sulfhydryl matrices to form covalently immobilized complexes that can subsequently be eluted with reducing agents. The interaction of GRCs with these matrices depends on the nature of both the immobilized sulfhydryl group and the type of leaving group attached. One matrix, agarose CL-4B-diaminoethyl-succinyl-thioethylamine-2-thiopyridyl+ ++ (DSTT), binds total receptor-bound steroid. A second matrix, agarose CL-4B-diaminoethyl-succinyl-cysteinyl-2-thiobenzoic acid (DSCT), binds activated but not nonactivated complexes. The reaction of activated complexes with the DSCT matrix is apparently through a sulfhydryl group located near the DNA binding domain, as soluble DNA interferes with the reaction. This sulfhydryl group(s) appears to be located in a portion of the GRC that is resistant to degradation, since proteolytic digestion of activated GRC to a point where DNA binding is lost results in only a moderate decrease in binding with the DSCT matrix. Purified receptor, covalently labeled with [3H]dexamethasone to the sulfhydryl associated with the steroid binding domain, was able to bind to DSCT matrix, providing evidence for distinct sulfhydryl groups associated with the steroid and DNA binding domains.     

Interaction of glucocorticoid receptor-steroid complexes with acceptor sites.

The binding of the "activated" receptor-glucocorticoid complexes of cultured rat hepatoma cells to nuclei, chromatin, and DNA has been studied under cell-free conditions. A critical factor in determining the shape of the binding curve is shown to be an inhibitory material which is present in crude cytosol and which can be removed without destroying the receptor-steroid complex. These and other results argue that the apparent saturation observed in earlier experiments may have been due to the inhibitors. Thus, the actual number of acceptor sites in hepatoma tissue culture cell nuclei is much larger than previously estimated and their affinity for the complex is lower. Nuclear binding experiments indicate that the inhibitory material interacts with the receptor-steroid complex. The inhibitors appear to be macromolecular; but their effects cannot be mimicked by albumin or hemoglobin. The acceptor capacity at low ionic strength for binding receptor-glucocorticoid complexes increases when proceeding from nuclei to DNA. An analysis of the kinetics of association and dissociation and of the relative binding behavior of nuclei and DNA argues that the affinity of complex for nuclei is much greater than for DNA. DNA-associated histones reduce the amount of complex that binds to DNA. These and perhaps other chromosomal proteins may be responsible for the ordering of acceptor capacity. Evidence is presented that the difference in affinities of nuclear and DNA acceptors could also be due to chromosomal proteins. In nuclei, these proteins may thus both reduce the amount of complex binding by rendering regions of DNA less accessible and increase the binding affinity of some, or all, of those DNA binding sites which remain exposed.     

Chromatin association and DNA binding properties of the c-fos proto-oncogene product.

As a first step in the analysis of the molecular function of the nuclear c-fos proto-oncogene product we have studied its subnuclear localization in serum-stimulated mouse fibroblasts where it forms a non-covalent, apparently monodisperse complex with another nuclear protein, p39. The c-fos/p39 complex is almost quantitatively released from intact nuclei by DNasel or micrococcus nuclease treatment under conditions where only a minor fraction of DNA and nuclear proteins is released. In gel filtration experiments, c-fos/p39 comigrates with chromatin and seems to be associated with regions of increased DNasel accessibility. c-fos/p39 is bound to chromatin by electrostatic forces of moderate strength since greater than 90% of the complex can be eluted from nuclei at 0.4 M NaCl. In vitro, the c-fos/p39 complex in nuclear extracts binds to double- and single-stranded calf thymus DNA, suggesting that the association of c-fos/p39 with chromatin is at least in part due to its interaction with DNA. In agreement with this conclusion, c-fos/p39 is released from nuclei by incubation with tRNA, presumably due to competition for binding sites. Our observations are compatible with the hypothesis that c-fos may play a role in the regulation of gene expression.     

The nuclear matrix is the site of glucocorticoid receptor complex action in the nucleus

Binding of highly purified glucocorticoid receptor complexes to nuclear matrix was evaluated. Extraction of purified nuclei with 2M potassium chloride and brief deoxyribonuclease digestion leaves a matrix structure containing 1% of nuclear DNA and 6-12% of nuclear proteins. The nuclear matrix retained two binding sites for receptor complexes, a high affinity, low capacity site and a low affinity, high capacity site. These sites have affinities and capacities consistent with those reported for binding of these complexes to intact nuclei. More extensive deoxyribonuclease treatment of the matrix resulted in a marked reduction of high affinity complex binding. Furthermore, the DNA binding form of the receptor complex but not the unactivated receptor complex bound to DNA fibers anchored to nuclear matrix as visualized by 18 nm gold particle receptor complexes. The data suggest that the nuclear matrix is the major site for coordinating glucocorticoid hormone action in the nucleus.     

Preferential binding of human XPA to the mitomycin C-DNA interstrand crosslink and modulation by arsenic and cadmium

The Xeroderma Pigmentosum A (XPA) protein is involved in the DNA damage recognition and repair complex formation steps of nucleotide excision repair (NER), and has been shown to preferentially bind to various forms of DNA damage including bulky lesions. DNA interstrand crosslinks are of particular interest as a form of DNA damage, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery, and mitomycin C (MMC) is one of several useful cancer chemotherapy drugs that induce these lesions. Purified XPA and the minimal DNA-binding domain of XPA are both fully capable of preferentially binding to MMC-DNA interstrand crosslinks in the absence of other proteins from the NER complex. Circular dichroism (CD) and gel shift assays were used to investigate XPA-DNA binding and to assess changes in secondary structure induced as a consequence of the interaction of XPA with model MMC-crosslinked and unmodified DNAs. These studies revealed that while XPA demonstrates only a modest increase in affinity for adducted DNA, it adopts a different conformation when bound to MMC-damaged DNA than when bound to undamaged DNA. This change in conformation may be more important in recruiting other proteins into a competent NER complex at damaged sites than preferential binding per se. Arsenic had little effect on XPA binding even at toxic concentrations, whereas cadmium reduced XPA binding to DNA to 10-15% that of Zn-XPA, and zinc addition could only partially restore activity. In addition, there was little or no change in conformation when Cd-XPA bound MMC-crosslinked DNA even though it demonstrated preferential binding, which may contribute to the mechanism by which cadmium can act as a co-mutagen and co-carcinogen.     

Activated steroid-receptor complex comparison of assays using DNA-cellulose or homologous nuclei

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [³H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the latter only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.     

Protein-induced bending of the simian virus 40 origin of replication

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.     

ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA.

Binding of adenosine (3-thiotriphosphate) (ATPgammaS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) was studied by filter binding. RFC alone bound 1.8 ATPgammaS molecules. However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPgammaS molecules, respectively, were bound. When both PCNA and DNA were present 3.6 ATPgammaS molecules were bound per RFC. Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA.PCNA.RFC complex. An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC.DNA complex dissociated with hydrolysis of ATP. Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC.ATP(2)); (ii) this complex binds PCNA (PCNA.RFC.ATP(2)), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA.RFC.ATP(3)); (iii) this complex can bind DNA to yield DNA.PCNA.RFC.ATP(3); (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA.PCNA.RFC.ATP(4) complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.     

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO₂ as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin‐mediated drug‐delivery of RGD‐modified DLDH (DLDH^RGD), leading to apoptotic cell death. In these experiments, DLDH^RGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA‐binding capability of this enzyme. To explore this interaction under cell‐free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel‐shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA‐binding. A Docking algorithm for prediction of protein‐DNA complexes, Paradoc, identified a putative DNA binding site at the C‐terminus of DLDH. Our finding that TiO₂‐bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.     

Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin

The plus-strand replication origin of bacteriophage fl has a bipartite structure consisting of a required core origin region and an adjacent A + T-rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein (gpII) and the enhancer binds the Escherichia coli integration host factor (IHF). gpII binds the core origin in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). We have used a double-label gel binding assay to determine the stoichiometry of the gpII-origin interaction. The results indicate that complex I contains two gpII molecules per origin, and complex II contains four gpII molecules per origin. Enhancer-independent mutations in gpII allow wild-type levels of replication in the absence of either the enhancer or IHF. We have examined the binding of an enhancer-independent gpII mutant (mp1) protein to the replication origin. The mp1 mutation in gpII (Met40----Ile) increases the co-operativity with which the protein binds to form complex II. In addition, the mutant gpII forms both complexes with a DNA fragment containing only two (beta-gamma) of the three repeats from the core origin sequence, while the wild-type protein forms only complex I with this fragment. We discuss how a mutation that increases the co-operativity of binding of an initiator protein might stimulate DNA replication.