DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos

In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos. We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins. In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo. This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.     

Participation of oligomerization through C-terminal D domain region of Sp1 in DNA binding

BACKGROUND: CINCA syndrome is a clinical syndrome of unclear etiology, characterized by a chronic multi-organ inflammatory process unsusceptible to treatment. CASE REPORT: An 18-month-old boy was admitted because he suffered, since the age of 2 months, from cutaneous, articular and neurological changes, lymphadenopathy, hepatosplenomegaly, choroiditis and psychosomatic development retardation. These clinical symptoms were unsusceptible to anti-inflammatory and antihistaminic drugs. Rehabilitation was not effective either. CONCLUSION: A long-term clinical observation is required before sustained multi-organ changes beginning in early childhood and exclusion of other chronic inflammatory diseases enable the diagnosis of CINCA syndrome.     

PCNA binding proteins in Drosophila melanogaster : the analysis of a conserved PCNA binding domain

The eukaryotic polymerase processivity factor, PCNA, interacts with cell cycle regulatory proteins such as p21(WAF1/Cip1) and Gadd45, as well as with proteins involved in the mechanics of DNA repair and replication. A conserved PCNA-binding motif is found in a subset of PCNA-interacting proteins, including p21, suggesting that the regulation of these interactions is important for the co-ordination of DNA replication and repair. We have identified several classes of protein which bind to Drosophila PCNA. Two of these proteins contain the consensus PCNA-binding domain: one is the Dacapo protein, a Drosophila homologue of p21(WAF1/Cip1), and the second is the transposase encoded by the Pogo DNA transposon . A conserved PCNA-binding domain is also present in a human relative of Pogo , named Tigger , suggesting that this domain has a functional role in this class of transposable element. This raises interesting possibilities for a novel method of transposition in which the transposase might be targeted to replicating DNA. Finally, we have investigated the use of this conserved PCNA-binding domain as a predictor of PCNA-binding capacity.     

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.     

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.     

Direct binding between two PDZ domain proteins Canoe and ZO-1 and their roles in regulation of the jun N-terminal kinase pathway in Drosophila morphogenesis

During Drosophila embryogenesis, the ventral epidermis dorsally expands and the left and right epithelial sheets meet and fuse along the dorsal midline. For this dorsal closure to occur, two PDZ domain proteins, Cno and ZO-1, are required. The dorsal epidermis remains open when the expression of ZO-1 and Cno are reduced simultaneously by hypomorphic mutations in the relevant loci. ZO-1 and Cno colocalize at adherens junctions in embryonic epithelia, and form a protein complex upon binding to each other. Genetic analysis showed that Cno is involved in the Jun N-terminal kinase (JNK) pathway for dorsal closure, as a modulator acting upstream of, or in parallel with, the small GTPase Drac1. The ZO-1-Cno complex may be involved in dynamic changes in cytoskeletal organization and cell adhesion during morphogenetic events associated with dorsal closure in the Drosophila embryo.     

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding

To examine a role for the medullary nucleus paragigantocellularis (PGi) in mediation of the symptomatology of opioid withdrawal, bilateral electrical stimulation of the PGi was performed in conscious, unrestrained, opioid naive (nondependent) rats. A characteristic series of behaviors was elicited during each 30-min session of PGi stimulation. The profile of these behaviors resembled qualitatively, but was not quantitatively identical with those seen during precipitated withdrawal from opioid dependence. This behavioral syndrome has been termed, opioid withdrawal-like behavior. The opioid withdrawal-like behaviors were voltage-, but not frequency-, dependent. Tolerance to repeated stimulation of the PGi did not develop following a series of 30-min runs of stimulation over 3.5 h. Intracerebroventricular (i.c.v.) injections of the nonselective opioid antagonist, naloxone, significantly decreased (by 40-50%) the intensity of stimulation-induced behavioral responses, as did injections of either the mu-selective (beta-funaltrexamine, beta-FNA) or the delta-selective (naltrindole, NTI) opioid antagonists. In contrast, similar i.c.v. injections of the kappa-selective antagonist, nor-binaltorphimine (nor-BNI), did not block behavioral responses to PGi stimulation. The results indicate that activation of the PGi by electrical stimulation can elicit behaviors similar to those observed during opioid withdrawal. Endogenous opioids, acting through mu- and delta-, but not kappa-opioid receptors, participate in mediating opioid withdrawal-like behaviors induced by PGi stimulation.     

Small abundant DNA binding proteins from the thermoacidophilic archaeon Sulfolobus shibatae constrain negative DNA supercoils

Major DNA binding proteins, designated Ssh7, were purified from the thermoacidophilic archaeon Sulfolobus shibatae. The Ssh7 proteins have an apparent molecular mass of 6.5 kDa and are similar to the 7-kDa DNA binding proteins from Sulfolobus acidocaldarius and Sulfolobus solfataricus in N-terminal amino acid sequence. The proteins constitute about 4.8% of the cellular protein. Upon binding to DNA, the Ssh7 proteins constrain negative supercoils. At the tested Ssh7/DNA mass ratios (0 to 1.65), one negative supercoil was taken up by approximately 20 Ssh7 molecules. Our results, together with the observation that the viral DNA isolated from S. shibatae is relaxed, suggest that regions of free DNA in the S. shibatae genome, if present, are highly positively supercoiled.     

DNA binding by cut homeodomain proteins is down-modulated by casein kinase II

Luminal Ag challenge of intestinal segments from sensitized rats results in a rapid (approximately 3 min) secretory response. We previously showed in horseradish peroxidase (HRP)-sensitized rats that the initial phase of transepithelial Ag transport occurred via a transcellular route and was enhanced by sensitization. However, following the hypersensitivity reaction, Ag also crossed between epithelial cells. The aim of this study was to determine the role of mast cells in the altered transepithelial Ag transport. White spotting mast cell-deficient rats and +/+ littermate controls were sensitized to HRP. After 10 to 14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. Electron microscopy of jejunum fixed at 2 min showed a similarly enhanced endocytic transport of HRP in sensitized +/+ and Ws/Ws rats compared with naive controls. In sensitized +/+ rats, a secretory response occurred approximately 3 min after challenge, and tissue conductance increased thereafter. Naive +/+ and sensitized Ws/Ws rats did not demonstrate a secretory response to HRP challenge, and conductance remained at baseline levels. The flux of HRP was elevated across tissue from sensitized +/+ rats but not across tissue from naive controls or sensitized Ws/Ws rats. The results indicate that sensitization enhances the initial phase of transepithelial uptake of Ag by transcytosis in a mast cell-independent manner. However, subsequent recruitment of the paracellular pathway for Ag transport in sensitized rats is dependent upon the presence of mast cells and occurs after the activation of such cells.     

Transition between different binding modes in rat DNA polymerase beta-ssDNA complexes

Interactions of rat DNA polymerase beta with a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence changes accompanying the binding, as a function of the thermodynamically rigorous binding density of rat pol beta-ssDNA complexes, reveals the existence of two binding phases. In the first high affinity phase, rat pol beta forms a complex with the ssDNA in which 16 nucleotides are occluded by the enzyme. In the second low affinity phase, a transition to a complex where the polymerase occludes only five nucleotides occurs. Thus, the data show that rat pol beta binds the ssDNA in two binding modes which differ in the number of occluded nucleotides. We designate the first complex as the (pol beta)16 binding mode and the second as the (pol beta)5 binding mode. The formation of the (pol beta)16 and (pol beta)5 modes has been fully confirmed in experiments with short ssDNA oligomers, a 16mer which can form either the (pol beta)16 or the (pol beta)5 mode, and a 10mer which can form only the (pol beta)5 mode. Binding of rat pol beta to the ssDNA has been analyzed using a statistical thermodynamic model which accounts for the existence of the two binding modes, cooperative interactions, and the overlap of potential binding sites. The results indicate that the 8 kDa domain of the enzyme is involved in ssDNA binding in both modes. Binding studies show that an isolated 8 kDa domain has the same intrinsic affinity for the ssDNA as the entire intact enzyme in its (pol beta)5 mode. However, the site size of the 8 kDa domain-ssDNA complex is ten nucleotides, suggesting that the formation of the (pol beta)5 mode is accompanied by a significant conformational transition of the intact protein. A higher intrinsic affinity, a higher net number of ions released, and a lower fluorescence change accompanying the formation of the (pol beta)16 than the (pol beta)5 mode indicate that the 31 kDa catalytic domain of the enzyme interacts with the ssDNA only in the (pol beta)16 mode. The significance of these results for understanding the functioning of rat pol beta in the DNA metabolism is discussed.