The V(D)J recombination signal sequence and kappa B binding protein Rc binds DNA as dimers and forms multimeric structures with its DNA ligands.

The murine DNA binding protein Rc binds to the heptamer motif of the V(D)J recombination signal sequences and to the kappa B motif of the immunoglobulin enhancer. Bacterial fusion proteins for Rc and DNA ligands of Rc form multiple protein-DNA complexes in electrophoretic mobility shift assays (EMSA). Large complexes formation is favored by an increased Rc concentration. In order to determine the architecture of these complexes, the apparent molecular weights of the protein-DNA complexes were first determined by their gel mobilities. The data suggest that Rc binds to its DNA ligands as dimers, tetramers, and multiples of tetramers. The inference that Rc binds DNA as dimers was substantiated by the formation of chimeric complexes when two electrophoretically distinguishable Rc proteins were employed in EMSA. Methylation interference experiments show that there are no contiguous protein binding sites evident in the DNA of the larger complexes. Apparently, multimerization occurs via protein-protein interactions. Such interaction was demonstrated by the formation of Rc dimers and tetramers in a chemical crosslinking experiment. Significantly, the multimerization of DNA-bound Rc could be involved in bringing the variable region gene segments together for the somatic V(D)J recombination.     

Mutations in two cysteine-histidine-rich clusters in adenovirus type 2 DNA polymerase affect DNA binding.

Several point and linker insertion mutations in two Cys-His-rich regions of adenovirus (Ad) DNA polymerase (Pol) gene have been expressed in recombinant vaccinia virus. The resulting mutant enzymes were analyzed in vitro for their effects on DNA synthesis activity, on Ad-specific initiation assays, on gel shifts of Ad origin sequences, and on interactions with adenovirus preterminal protein (pTP) and nuclear factor I (NFI). In general, mutants in downstream Cys-His sequences had a pronounced effect in these assays. Mutants in the upstream Cys-His region had a moderate effect on DNA synthesis and elongation but failed to make dCMP-pTP initiation complexes and failed to make specific shifted complexes in a gel retardation assay. These mutants could still bind to pTP and NFI in a coimmunoprecipitation experiment, suggesting that this upstream Cys-His region of Ad Pol is involved either in specific Ad DNA origin binding or in nonspecific DNA binding. Changing residues within Cys doublets in the downstream Cys-His region had pronounced effects on many Ad Pol functions such as DNA synthesis, DNA binding, and in vitro initiation; however, these mutants showed little reduction in binding to pTP and NFI; mutants at other cysteines or histidines within this region of Ad Pol did not appear to have an effect on enzyme function. This observation suggests that the downstream Cys-His region of Ad Pol is important for DNA binding and might fold into a Zn finger motif.     

Secondary structures in CpG oligonucleotides affect immunostimulatory activity

Oligodeoxynucleotides containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system. Our previous studies showed that the 5(')-end of a CpG oligonucleotide should be accessible for receptor recognition and subsequent immune stimulation. Activity is abrogated if this end is blocked by joining two CpG oligos through 5(')-5(') linkage. It was not known whether a similar effect would arise from secondary structures at either end of a CpG oligo, such as hairpin loops or terminal dimers. In the present study we found that 5(')-terminal secondary structures affect activity significantly more than those at the 3(')-end. The need for an open 5(')-end suggests that the receptor responsible for immune stimulation reads the DNA sequence from this end. These results may also provide insights to place CpG motifs appropriately in DNA vaccines to induce additional Th1 type responses.     

Loop lengths of G-quadruplex structures affect the G-quadruplex DNA binding selectivity of the RGG motif in Ewing's sarcoma

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro. Here, we show that the RGG of EWS binds preferentially to G-quadruplexes with longer loops, which is not related to the topology of the G-quadruplex structure. Moreover, the G-quadruplex DNA binding of the RGG in EWS depends on the phosphate backbone of the loops in the G-quadruplex DNA. We also investigated the G-quadruplex DNA binding activity of the N- and C-terminally truncated RGG to assess the role of the regions in the RGG in G-quadruplex DNA binding. Our findings indicate that the RGG and the other arginine-rich motif of residues 617-656 of the RGG in EWS are important for the specific binding to G-quadruplex DNA. These findings will contribute to the development of molecules that selectively target different G-quadruplex DNA.     

Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI.     

Differential binding of quadruplex structures of muscle-specific genes regulatory sequences by MyoD, MRF4 and myogenin

Four myogenic regulatory factors (MRFs); MyoD, Myf-5, MRF4 and Myogenin direct muscle tissue differentiation. Heterodimers of MRFs with E-proteins activate muscle-specific gene expression by binding to E-box motifs d(CANNTG) in their promoters or enhancers. We showed previously that in contrast to the favored binding of E-box by MyoD-E47 heterodimers, homodimeric MyoD associated preferentially with quadruplex structures of regulatory sequences of muscle-specific genes. To inquire whether other MRFs shared the DNA binding preferences of MyoD, the DNA affinities of hetero- and homo-dimeric MyoD, MRF4 and Myogenin were compared. Similarly to MyoD, heterodimers with E47 of MRF4 or Myogenin bound E-box more tightly than quadruplex DNA. However, unlike homodimeric MyoD or MRF4, Myogenin homodimers associated weakly and nonpreferentially with quadruplex DNA. By reciprocally switching basic regions between MyoD and Myogenin we demonstrated dominance of MyoD in determining the quadruplex DNA-binding affinity. Thus, Myogenin with an implanted MyoD basic region bound quadruplex DNA nearly as tightly as MyoD. However, a grafted Myogenin basic region did not diminish the high affinity of homodimeric MyoD for quadruplex DNA. We speculate that the dissimilar interaction of MyoD and Myogenin with tetrahelical domains in muscle gene promoters may differently regulate their myogenic activities.     

Kinetics of actin-myosin binding. ATP-ase activity and sol-gel dissipative structures

The simple kinetic model of actin-myosin binding-dissociation process including ATP-ase activity is considered. We demonstrated how one may easily include cooperativity in such a model by using effectivity factors introduced in our previous papers. A possibility of further simplifying the model through quasi-stationary approximation for some variables is considered. Sol-gel dissipative structures and possible biological implications of such structures are discussed.     

RecG helicase activity at three- and four-strand DNA structures.

The RecG helicase of Escherichia coli is necessary for efficient recombination and repair of DNA in vivo and has been shown to catalyse the unwinding of DNA junctions in vitro. Despite these findings, the precise role of RecG remains elusive. However, models have been proposed in which RecG promotes the resolution of linked duplexes by targeting three-strand junctions present at D-loops. One such model postulates that RecG catalyses the formation of four-strand (Holliday) junctions from three-strand junctions. To test this model, the DNA binding and unwinding activities of RecG were analysed using synthetic three- and four-strand junctions. The substrate specificity of RecG was found to depend critically on the concentrations of ATP and MgCl(2)and under certain conditions RecG preferentially unwound three-strand junction DNA. This was at least partly due to the larger inhibitory effect of MgCl(2)on the binding of four-strand as opposed to three-strand junctions by RecG. Thus RecG may be targeted to three-strand junctions in vivo whilst still being able to branch migrate the four-strand junctions formed as a result of the initial helicase reaction. The increase in the dissociation constant of RecG on conversion of a three-strand into a four-strand junction may also facilitate resolution of the four-strand junction by the RuvABC complex.     

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.     

Lac repressor purification without inactivation of DNA binding activity.

A procedure has been developed which eliminates the commonly observed inactivation of the DNA binding activity of the lac repressor during purification. The operator binding activity of the repressor obtained by this method is 100 +/- 10%. The repressor can be stored frozen indefinitely without losing its affinity for DNA.     

Polymorphisms in human langerin affect stability and sugar binding activity

Langerhans cells are specialized skin dendritic cells that take up and degrade antigens for presentation to the immune system. Langerin, a cell surface C-type lectin of Langerhans cells, can be internalized and accumulates in Birbeck granules, subdomains of the endosomal recycling compartment that are specific to Langerhans cells. Langerin binds and mediates uptake and degradation of glycoconjugates containing mannose and related sugars. Analysis of the human genome has identified three single nucleotide polymorphisms that result in amino acid changes in the carbohydrate-recognition domain of langerin. The effects of the amino acid changes on the activity of langerin were examined by expressing each of the polymorphic forms. Expression of full-length versions of the four common langerin haplotypes in fibroblasts revealed that all of these forms can mediate endocytosis of neoglycoprotein ligands. However, sugar binding assays and differential scanning calorimetry performed on fragments from the extracellular domain showed that two of the amino acid changes reduce the affinity of the carbohydrate-recognition domain for mannose and decrease the stability of the extracellular domain. In addition, analysis of sugar binding by langerin containing the rare W264R mutation, previously identified in an individual lacking Birbeck granules, shows that this mutation abolishes sugar binding activity. These findings suggest that certain langerin haplotypes may differ in their binding to pathogens and thus might be associated with susceptibility to infection.     

Specific binding of cruciform DNA structures by a protein from human extracts.

A gel electrophoresis binding assay has been used to probe extracts from cultured human lymphoblasts for proteins that bind cruciform structures in duplex DNA. Proteins have been detected that form complexes with synthetic X- and Y-junctions. Several lines of evidence suggest that binding is specific for DNA structure rather than sequence: (1) X- and Y-structures were bound whereas linear duplexes containing identical DNA sequences were not, (2) Binding occurred with equal efficiency to two X-junctions that were constructed from DNA strands of different sequence, (3) One X-junction successfully competed with another for binding whereas linear duplex DNA did not; and (4) protein-DNA complexes were observed at probe:non-specific competitor DNA ratios of 1:10,000.     

Human adenovirus proteinase: DNA binding and stimulation of proteinase activity by DNA.

The interaction of the human adenovirus proteinase (AVP) with various DNAs was characterized. AVP requires two cofactors for maximal activity, the 11-amino acid residue peptide from the C-terminus of adenovirus precursor protein pVI (pVIc) and the viral DNA. DNA binding was monitored by changes in enzyme activity or by fluorescence anisotropy. The equilibrium dissociation constants for the binding of AVP and AVP-pVIc complexes to 12-mer double-stranded (ds) DNA were 63 and 2.9 nM, respectively. DNA binding was not sequence specific; the stoichiometry of binding was proportional to the length of the DNA. Three molecules of the AVP-pVIc complex bound to 18-mer dsDNA and six molecules to 36-mer dsDNA. When AVP-pVIc complexes bound to 12-mer dsDNA, two sodium ions were displaced from the DNA. A Delta of -4.6 kcal for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy results from nonspecific interactions between the AVP-pVIc complex and DNA. The cofactors altered the interaction of the enzyme with the fluorogenic substrate (Leu-Arg-Gly-Gly-NH)2-rhodamine. In the absence of any cofactor, the Km was 94.8 microM and the kcat was 0.002 s(-1). In the presence of adenovirus DNA, the Km decreased 10-fold and the kcat increased 11-fold. In the presence of pVIc, the Km decreased 10-fold and the kcat increased 118-fold. With both cofactors present, the kcat/Km ratio increased 34000-fold, compared to that with AVP alone. Binding to DNA was coincident with stimulation of proteinase activity by DNA. Although other proteinases have been shown to bind to DNA, stimulation of proteinase activity by DNA is unprecedented. A model is presented suggesting that AVP moves along the viral DNA looking for precursor protein cleavage sites much like RNA polymerase moves along DNA looking for a promoter.     

Photodynamic activity of dyes with different DNA binding properties I. free-radical induction in DNA.

The photosensitizing efficiencies of eight dyes have been compared; two acridines, two xanthene derivatives, one sulphur-containing dye and three chemotherapeutic agents. The analysed reaction was the photosensitized induction of free radicals in calf-thymus DNA at low temperature. The binding of these dyes to DNA was first measured. Both strong (process I) and weak (process II) binding, with different intensities, either alone or together, were observed as mode of fixation. Whatever the nature of their binding, all the dyes used revealed a photosensitizing power as inducers of peroxide radicals in DNA. Their relative efficiencies, expressed as a function of the amount of dye molecules bound to DNA, were found to be very different. Intercalation, however, appeared to favour the free-radical induction as the first strongly bound molecules were more efficient.     

DNA binding activity of the BarH1 homeodomain of Drosophila.

Using a series of deletion mutants of BarH1, a Drosophila homeobox gene required for eye morphogenesis, the DNA-binding region of the BarH1 protein was determined. Not only homeodomain but also its upstream sequence were found to be necessary for binding, whereas about a half of the conserved downstream sequence (Bar domain) was dispensable.