Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha.

The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.

Dissection of transcription factor TFIIF functional domains required for initiation and elongation.

TFIIF is unique among the general transcription factors because of its ability to control the activity of RNA polymerase II at both the initiation and elongation stages of transcription. Mammalian TFIIF, a heterodimer of approximately 30-kDa (RAP30) and approximately 70-kDa (RAP74) subunits, assists TFIIB in recruiting RNA polymerase II into the preinitiation complex and activates the overall rate of RNA chain elongation by suppressing transient pausing by polymerase at many sites on DNA templates. A major objective of efforts to understand how TFIIF regulates transcription has been to establish the relationship between its initiation and elongation activities. Here we establish this relationship by demonstrating that TFIIF transcriptional activities are mediated by separable functional domains. To accomplish this, we sought and identified distinct classes of RAP30 mutations that selectively block TFIIF activity in transcription initiation and elongation. We propose that (i) TFIIF initiation activity is mediated at least in part by RAP30 C-terminal sequences that include a cryptic DNA-binding domain similar to conserved region 4 of bacterial sigma factors and (ii) TFIIF elongation activity is mediated in part by RAP30 sequences located immediately upstream of the C terminus in a region proposed to bind RNA polymerase II and by additional sequences located in the RAP30 N terminus.

The Ran/TC4 GTPase-binding domain: identification by expression cloning and characterization of a conserved sequence motif.

Ran/TC4 is an essential, nuclear GTPase implicated in the initiation of DNA replication, entry into and exit from mitosis, and in nuclear RNA and protein transport through the nuclear pore complex. This diversity of functions suggests that Ran interacts with a large number of down-stream targets. Using an overlay assay, we detected a family of putative target proteins that associate with GTP-bound Ran. The sequence of only one such protein, HTF9a/RanBP1, is known. We have now cloned two additional Ran-binding proteins, allowing identification of a distinctive, highly conserved sequence motif of approximately 150 residues. This motif represents a minimal Ran-binding domain that stabilizes the GTP-bound state of Ran. The isolated domain also functions as a coactivator of Ran-GTPase-activating protein. Mutation of a conserved residue within the Ran-binding domain of HTF9a protein drastically reduced Ran binding. Ran-binding proteins coimmunoprecipitated with epitope-tagged Ran from cell lysates, suggesting that these proteins may associate in vivo. A previously uncharacterized Caenorhabditis elegans gene could encode a protein (96 kDa) possessing two Ran-binding domains. This open reading frame also contains similarities to nucleoporins, suggesting a functional link between Ran and nuclear pore complexes.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

The Structure of theCyprinid herpesvirus 3ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.

Zalpha-domains: At the intersection between RNA editing and innate immunity

The involvement of A to I RNA editing in antiviral responses was first indicated by the observation of genomic hyper-mutation for several RNA viruses in the course of persistent infections. However, in only a few cases an antiviral role was ever demonstrated and surprisingly, it turns out that ADARs - the RNA editing enzymes - may have a prominent pro-viral role through the modulation/down-regulation of the interferon response. A key role in this regulatory function of RNA editing is played by ADAR1, an interferon inducible RNA editing enzyme. A distinguishing feature of ADAR1, when compared with other ADARs, is the presence of a Z-DNA binding domain, Zalpha. Since the initial discovery of the specific and high affinity binding of Zalpha to CpG repeats in a left-handed helical conformation, other proteins, all related to the interferon response pathway, were shown to have similar domains throughout the vertebrate lineage. What is the biological function of this domain family remains unclear but a significant body of work provides pieces of a puzzle that points to an important role of Zalpha domains in the recognition of foreign nucleic acids in the cytoplasm by the innate immune system. Here we will provide an overview of our knowledge on ADAR1 function in interferon response with emphasis on Zalpha domains.

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-I chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-gamma and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-gamma responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N76-114, each of which contained nonameric H2(d) binding domains with high binding scores for both class I and, except for N76-93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-gamma and IL-4 responses and strong memory CTL responses to the LAMP-N chimera. (C) 2005 Elsevier Inc. All rights reserved.

Binding of small molecules to DNA junctions

Previous results in our laboratory suggest that the (CG) 4 segments whether present in a right-handed or a left-handed conformation form distinctive junctions with adjacent random sequences. These junctions and their associated sequences have unique structural and thermodynamic properties that may be recognized by DNA-binding molecules. This study probes these sequences by using the following small ligands: actinomycin D, 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione, ametantrone, and tris(phenanthroline)ruthenium (II). These ligands may recognize the distinctive features associated to the (CG)4 segment and its junctions and thus interact preferentially near these sequences. Restriction enzyme inhibition assays were used to determine whether or not binding interactions took place, and to approximate locations of these interactions. These binding studies are first carried out using two small synthetic oligomers BZ-III and BZ-IV. The (5meCG)4 segment present in BZ-III adopts the Z-conformation in the presence of 50 m M Co(NH3)63+. In BZ-IV, the unmethylated (CG)4 segment changes to a non-B conformation in the presence of 50 m M Co(NH3)63+. BZ-IV, containing the (CG)4 segment, was inserted into a clone plasmid then digested with the restriction enzyme Hinf I to produce a larger fragment that contains the (CG)4 segment. The results obtained on the small oligomers and on the larger fragment for restriction enzyme Mbo I indicate that 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione binds more efficiently at or near the (CG)4 segment. Restriction enzymes EcoRV, Sac I and Not I with cleavage sites upstream and downstream of the (CG)4 insert were used to further localize binding interactions in the vicinity of the (CG)4 insert. RNA polymerase activity was studied in a plasmid which contained the (CG)4 insert downstream from the promoter sites of SP6 and T7 RNA polymerases. Activities of these two polymerases were studied in the presence of each one of the ligands used throughout the study. Only actinomycin D and spider, which bind at or near the (CG)4 segment, alter the activities of SP6 and T7 RNA polymerases. Surprisingly, enhancement of polymerase activity was observed in the presence of very low concentrations of actinomycin D. These results suggest that the conformational features of (CG) segments may serve in regulatory functions of DNA. ^

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Dissecting the Relation between a Nuclear Receptor and GATA: Binding Affinity Studies of Thyroid Hormone Receptor and GATA2 on TSH beta Promoter

Background: Much is known about how genes regulated by nuclear receptors (NRs) are switched on in the presence of a ligand. However, the molecular mechanism for gene down-regulation by liganded NRs remains a conundrum. The interaction between two zinc-finger transcription factors, Nuclear Receptor and GATA, was described almost a decade ago as a strategy adopted by the cell to up-or down-regulate gene expression. More recently, cell-based assays have shown that the Zn-finger region of GATA2 (GATA2-Zf) has an important role in down-regulation of the thyrotropin gene (TSH beta) by liganded thyroid hormone receptor (TR). Methodology/Principal Findings: In an effort to better understand the mechanism that drives TSH beta down-regulation by a liganded TR and GATA2, we have carried out equilibrium binding assays using fluorescence anisotropy to study the interaction of recombinant TR and GATA2-Zf with regulatory elements present in the TSH beta promoter. Surprisingly, we observed that ligand (T3) weakens TR binding to a negative regulatory element (NRE) present in the TSH beta promoter. We also show that TR may interact with GATA2-Zf in the absence of ligand, but T3 is crucial for increasing the affinity of this complex for different GATA response elements (GATA-REs). Importantly, these results indicate that TR complex formation enhances DNA binding of the TR-GATA2 in a ligand-dependent manner. Conclusions: Our findings extend previous results obtained in vivo, further improving our understanding of how liganded nuclear receptors down-regulate gene transcription, with the cooperative binding of transcription factors to DNA forming the core of this process.

Involvement of the N-finger in the self-association of GATA-1

Zinc fingers are recognized as small protein domains that bind to specific DNA sequences. Recently however, zinc fingers from a number of proteins, in particular the GATA family of transcription factors, have also been implicated in specific protein-protein interactions. The erythroid protein GATA-1 contains two zinc fingers: the C-finger, which is sufficient for sequence-specific DNA-binding, and the N-finger, which appears both to modulate DNA-binding and to interact with other transcription factors. We have expressed and purified the N-finger domain and investigated its involvement in the self-association of GATA-1. We demonstrate that this domain does not homodimerize but instead makes intermolecular contacts with the C-finger, suggesting that GATA dimers are maintained by reciprocal N-finger-C-finger contacts. Deletion analysis identifies a 25-residue region, C-terminal to the core N-finger domain, that is sufficient for interaction with intact GATA-1. A similar subdomain exists C-terminal to the C-finger, and we show that self-association is substantially reduced when both subdomains are disrupted by mutation. Moreover, mutations that impair GATA-1 self-association also interfere with its ability to activate transcription in transfection studies.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Characterization of the sequential non-covalent and covalent interactions of the antitumour antibiotic hedamycin with double stranded DNA by NMR spectroscopy

Hedamycin, a member of the pluramycin class of antitumour antibiotics, consists of a planar anthrapyrantrione chromophore to which is attached two aminosugar rings at one end and a bisepoxide-containing sidechain at the other end, Binding to double-stranded DNA is known to involve both reversible and non-reversible modes of interaction. As a part of studies directed towards elucidating the structural basis for the observed 5'-pyGT-3' sequence selectivity of hedamycin, we conducted one-dimensional NMR titration experiments at low temperature using the hexadeoxyribonucleotide duplexes d(CACGTG)(2) and d(CGTACG)(2). Spectral changes which occurred during these titrations are consistent with hedamycin initially forming a reversible complex in slow exchange on the NMR timescale and binding through intercalation of the chromophore. Monitoring of this reversible complex over a period of hours revealed a second type of spectral change which corresponds with formation of a non-reversible complex. Copyright (C) 1999 John Wiley & Sons, Ltd.