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PubMed1994

HU and functional analogs in eukaryotes promote Hin invertasome assembly.

The prokaryotic protein HU functions as an accessory factor in many different biochemical reactions. We have characterized the role of HU in assembling the invertasome, an intermediate nucleoprotein complex involved in Hin-mediated site-specific recombination. Formation of this complex requires the looping of intervening DNA segments between sites bound by the Hin recombinase and the Fis protein. HU stimulates this process on substrates containing intervening segments of length < 100 bp. Characterization of the activity of HU in Hin-mediated recombination in vitro and in vivo yields evidence that its role in this reaction is primarily to facilitate the looping of the intervening DNA segment. By using this reaction as an assay, we identify proteins from mammals, yeast, trypanosomes, and wheat which can fulfill the same function in vitro. Using ligase-mediated circularization of short DNA fragments we also show that HU, the high mobility group (HMG) 1 and 2 proteins from mammals, and a protein from yeast can bend DNA extremely efficiently. These results support the view that this ubiquitous class of proteins enhance the assembly of nucleoprotein complexes under conditions of limited DNA flexibility.

Animals↗
PubMed1989

Alterations in the polypyrimidine sequence affect the in vitro splicing reactions catalyzed by HeLa cell-free preparations.

The polypyrimidine tract, located at the 3' end of intron 1 of the adenovirus major late transcript, was studied for its role in splicing using cell-free preparations isolated from HeLa cells. A plasmid (pIz) was constructed in which seven purine bases were substituted for pyrimidine bases within the 14-nucleotide polypyrimidine sequence. Runoff transcripts extending to the middle of intron 2 were tested for their ability to support in vitro splicing. The efficiency of these reactions was compared with pre-mRNA transcripts made from the wild-type nonmutated plasmid (p1-2). Neither spliced products nor splicing intermediates were detected in reactions with the pIz pre-mRNA. The formation of the nucleoprotein complexes involved in splicing was examined with this altered pre-mRNA. No 55 S splicing complex was detected and only low levels of the 30 S presplicing complex formed (30-fold less than with wild-type pre-mRNA). However, when a longer runoff transcript was prepared from the polypyrimidine mutated plasmid pIz, spliced RNA was formed. This activity required specific downstream sequences, since transcripts produced from pIz which contained substituted downstream sequences were not spliced. Although intron 2 of the adenovirus major late transcript does not contain a discernible 3' polypyrimidine sequence, pre-mRNA (p2-3) containing this intron was efficiently spliced. However, when the 3' region of intron 2 was substituted for the polypyrimidine sequence of intron 1, the resulting pre-mRNA did not support efficient splicing in vitro. However, when the polypyrimidine sequence of intron 1 was substituted for the sequence at the 3' end of intron 2, efficient splicing occurred, and the rate of formation of splicing intermediates and the accumulation of nucleoprotein complexes was greater than with the wild-type pre-mRNA (p2-3).

Adenoviruses, Human↗
PubMed1998

Human T-cell leukemia virus type 1 Tax requires direct access to DNA for recruitment of CREB binding protein to the viral promoter.

Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.

Binding Sites↗
PubMed2006

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

Animals↗
PubMed2006

Analysis of in-vivo LacR-mediated gene repression based on the mechanics of DNA looping.

Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58-156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes.

Base Sequence↗
PubMed2005

Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex.

Polycomb response elements (PREs) are cis-acting DNA elements that mediate epigenetic gene silencing by Polycomb group (PcG) proteins. Here, we report that Pleiohomeotic (PHO) and a multiprotein Polycomb core complex (PCC) bind highly cooperatively to PREs. We identified a conserved sequence motif, named PCC-binding element (PBE), which is required for PcG silencing in vivo. PHO sites and PBEs function as an integrated DNA platform for the synergistic assembly of a repressive PHO/PCC complex. We termed this nucleoprotein complex silenceosome to reflect that the molecular principles underpinning its assemblage are surprisingly similar to those that make an enhanceosome.

Animals↗
PubMed1996

The crystal structure of the DNA-binding domain of yeast RAP1 in complex with telomeric DNA.

Telomeres, the nucleoprotein complexes at the ends of eukaryotic chromosomes, are essential for chromosome stability. In the yeast S. cerevisiae, telomeric DNA is bound in a sequence-specific manner by RAP1, a multifunctional protein also involved in transcriptional regulation. Here we report the crystal structure of the DNA-binding domain of RAP1 in complex with telomeric DNA site at 2.25 A resolution. The protein contains two similar domains that bind DNA in a tandem orientation, recognizing a tandemly repeated DNA sequence. The domains are structurally related to the homeodomain and the proto-oncogene Myb, but show novel features in their DNA-binding mode. A structured linker between the domains and a long C-terminal tail contribute to the binding specificity. This structure provides insight into the recognition of the conserved telomeric DNA sequences by a protein.

Amino Acid Sequence↗
PubMed2001

A DNA polymerase epsilon inhibitor activates the ribo and deoxyribo modes of primase expression and induces a unique phenomenon of primer accumulation.

Carbonyldiphosphonate (COMDP), a selective inhibitor of DNA polymerase (pol) epsilon, strongly stimulates expression of the ribo and deoxyribo modes of primase (Pr) activities of the Pr-DNA pol alpha enzyme complex associated with special cytoplasmic nucleoprotein complexes of chicken leukemic myeloblasts [J. Ríman and A. Sulová, Acta Virol. 41 (1997) 181-214]. Besides stimulation, COMDP uncouples the Pr activities from those of DNA pol alpha, inducing in this way a unique phenomenon of accumulation of primers of basic length. In the presence of dNTPs, the COMDP effect is counteracted by excess of mimosine. The mutually exclusive effects of these agents are discussed.

DNA Polymerase I↗
PubMed1995

Mutual stabilisation of bacteriophage Mu repressor and histone-like proteins in a nucleoprotein structure.

Integration host factor (IHF) binds in a sequence-specific manner to the bacteriophage Mu early operator. It participates with bound Mu repressor, c, in building stable, large molecular mass nucleoprotein complexes in vitro and enhances repression of early transcription in vivo. We demonstrate that, when the specific IHF binding site with the operator is mutated, the appearance of large molecular mass complexes still depends on IHF and c, but the efficiency of their formation is reduced. Moreover, the IHF-like HU protein, which binds DNA in a non-sequence-specific way, can substitute for IHF and participate in complex formation. Since the complexes require both c and a host factor (IHF or HU), the results imply that these proteins stabilise each other within the nucleoprotein structures. These results suggest that IHF and HU are directed to the repressor-operator complexes, even in the absence of detectable sequence-specific binding. This could be a consequence of their preferential recognition of DNA containing a distortion such as that introduced by repressor binding to the operator. The histone-like proteins could then stabilise the nucleoprotein complexes simply by their capacity to maintain a bend in DNA rather than by specific protein-protein interactions with c. This model is supported by the observation that the unrelated eukaryotic HMG-1 protein, which exhibits a similar marked preference for structurally deformed DNA, is also able to participate in the formation of higher-order complexes with c and the operator DNA.

Animals↗
PubMed2001

Dynamics of Fos-Jun-NFAT1 complexes.

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes.

Allosteric Regulation↗
PubMed2003

A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament.

Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.

Adenosine Triphosphatases↗
PubMed2005

Gal repressor-operator-HU ternary complex: pathway of repressosome formation.

DNA transaction reactions require formation of nucleoprotein complexes that involve multifaceted DNA-protein and protein-protein interactions. Genetic and biochemical studies suggested that the higher order Gal repressosome structure, which governs the transcription of two tandem galpromoters in Escherichia coli, involves sequence-specific binding of GalR repressor dimers to two operators, O(E) and O(I), located 113 bp apart, binding of GalR to the sequence-nonspecific DNA binding protein HU, interaction of HU with an architecturally critical DNA site between the two operators, and interaction between two DNA-bound GalR dimers generating a loop of the intervening DNA segment. In this paper, we demonstrate and determine the thermodynamic parameters of several of these interactions, GalR dimer-O(E), GalR tetramerization, HU-GalR, and HU-GalR-O(E) interactions, by analytical ultracentrifugation, fluorescence anisotropy, and fluorescence resonance energy transfer. The physiological significance of several of these interactions was confirmed by the finding that a mutant HU, which is unable to form the repressosome in vivo and in vitro, failed to show the HU-GalR interaction. The results help to construct a pathway of Gal repressosome assembly.

Base Sequence↗
PubMed1997

Anchoring of CREB binding protein to the human T-cell leukemia virus type 1 promoter: a molecular mechanism of Tax transactivation.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates viral transcription through interaction with the cellular transcription factor CREB (cyclic AMP response element [CRE] binding protein). Although Tax stabilizes the binding of CREB to the Tax-responsive viral CREs in the HTLV-1 promoter, the precise molecular mechanism by which Tax mediates strong transcriptional activation through CREB remains unclear. In this report, we show that Tax promotes high-affinity binding of the KIX domain of CREB binding protein (CBP) to CREB-viral CRE complexes, increasing the stability of KIX in these nucleoprotein complexes by up to 4.4 kcal/mol. Comparable KIX binding affinities were measured for both phosphorylated and unphosphorylated forms of CREB, and in all cases high-affinity binding was dependent upon both Tax and the viral CRE. Tax also promoted association of KIX to a truncated form of CREB containing only the 73-amino-acid basic leucine zipper (bZIP) domain, indicating that the entire amino-terminal CBP-interacting domain of CREB is nonessential in the presence of Tax. Functional studies upheld the binding studies, as expression of the bZIP domain of CREB was sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Finally, we show that transfection of a KIX expression plasmid, which lacks activation properties, inhibited Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely a cofactor in mediating Tax stimulation of HTLV-1 transcription. Together, these data support a model in which Tax anchors CBP to the HTLV-1 promoter, with strong transcriptional activation resulting from the CBP-associated activities of nucleosome remodeling and recruitment of the general transcription machinery.

Activating Transcription Factor 1↗
PubMed2000

Stoichiometry of P1 plasmid partition complexes.

The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1. We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.

Bacterial Proteins↗
PubMed1999

Nuclear import of human immunodeficiency virus type-1 preintegration complexes.

Following infection-mediated entry into the cytoplasm, retroviral cores form large nucleoprotein complexes (PICs) which undergo reverse transcription and, ultimately, catalyze provirus formation. The ability of these complexes to be specifically imported into the nucleus via NPCs explains why nondividing cells can be productively infected with lentiviruses such as HIV-1, whereas productive infection by the oncoretrovirus MLV is restricted to proliferating cells. Current evidence suggests that virally encoded protein components of the HIV-1 PIC, in particular IN and Vpr, act in concert to target these complexes for nuclear import by recruiting cellular import factors and interacting with the NPCs. Here have we reviewed recent advances made in this complex and fascinating area of HIV-1 biology and have discussed them in relation to models for postentry nuclear import in other retroviral and nonretroviral systems.

Adenoviridae↗
PubMed1983

Pf1 bacteriophage replication--assembly complex. X-ray fibre diffraction of the high humidity form.

The helical intracellular nucleoprotein complex of Pf1 bacteriophage has been studied by X-ray fibre diffraction in various hydration states. The helix pitch changes from 44 A in dry fibres to 55 A in wet fibres, whereas the unit rise between subunits in the helix apparently does not change with humidity. This result indicates that the nucleoprotein assembly twists more readily than it stretches. This is consistent with its biological role of tightening the viral DNA into a more compact form for packaging in the virion.

Coliphages↗
PubMed1996

Atomic force microscopy of DNA, nucleoproteins and cellular complexes: the use of functionalized substrates.

Progress towards rapid and simple characterization of biomolecular samples by scanning probe microscopy is impeded mainly by limitations of the current approach to sample preparation. We are working on approaches based on chemical functionalization of mica. Treatment of mica with aminopropyltriethoxy silane (APTES) makes the surface positively charged (AP-mica) and able to hold DNA in place for imaging, even in water. We have shown that AP-mica is an appropriate substrate for numerous nucleoprotein complexes as well. The AFM images of the complex of DNA with RecA protein are stable and indicate a structural periodicity for this filament. AP-mica holds strongly such large DNA complexes as kinetoplast DNA (kDNA) and is an appropriate substrate for their imaging with AFM. We have further develop this approach for making hydrophobic substrates. Silylation of mica surface with hexamethyldisilazane (Me-mica) allowed us to get AFM images of chlorosomes, an antenna complex isolated from green photosynthetic bacteria. Me-mica may be converted into a positively charged substrate after treatment with water solutions of tetraethylammonium bromide or cetyltrimethylammonium bromide. These activated surfaces show high activity towards binding the DNA molecules.

Aluminum Silicates↗