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PubMed2001

The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex.

Stable maintenance of P1 plasmids in Escherichia coli is mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and the E. coli integration host factor (IHF) bound specifically to the P1 parS site. IHF strongly stimulates ParB binding to parS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex. A fragment missing only the last 16 amino acids of ParB bound specifically to parS, but binding was weak and was no longer stimulated by IHF. The ability of IHF to stimulate ParB binding to parS correlated with the ability of ParB to dimerize via its C terminus. Using full and partial parS sites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences within parS. Based on these data, we have proposed a model of how the ParB dimer binds parS to form the minimal partition complex.

Bacterial Proteins↗

PubMed1999

The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin.

Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P). G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P. G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution. G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P. Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture. The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin. Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate. G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex. We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication.

Adenosine Triphosphate↗

PubMed2000

Characterization of a Tn5 pre-cleavage synaptic complex.

Protein catalyzed DNA rearrangements typically require assembly of complex nucleoprotein structures. In transposition and integration reactions, these structures, termed synaptic complexes, are mandatory for catalysis. We characterize the Tn5 pre-cleavage synaptic complex, the simplest transposition complex described to date. We identified this complex by gel retardation assay using short, linear fragments and have shown that it contains a dimer of transposase, two DNA molecules, and is competent for DNA cleavage in the presence of Mg(2+). We also used hydroxyl radical footprinting and interference techniques to delineate the protein-DNA contacts made in the Tn5 synaptic and monomer complexes. All positions (except position 1) of the end sequence are contacted by transposase in the synaptic complex. We have determined that positions 2-5 of the end sequence are specifically required for synaptic complex formation as they are not required for monomer complex formation. In addition, in the synaptic complex, there is a strong, local distortion centered around position 1 which likely facilitates cleavage.

Bacteria↗

PubMed2001

Long-range electrostatic interactions influence the orientation of Fos-Jun binding at AP-1 sites.

Heterodimeric transcription regulatory proteins that bind palindromic DNA sequences can potentially bind their recognition sites in two opposite orientations. The orientation of transcription factor binding can control transcriptional activity by altering interactions with proteins that bind to adjacent regulatory elements. Fos-Jun heterodimers bind to AP-1 sites with different flanking sequences in opposite orientations. A gel-based fluorescence resonance energy transfer assay, gelFRET, was used to define the mechanism whereby amino acid residues and nucleotide base-pairs outside the Fos-Jun-AP-1 contact interface determine the orientation of heterodimer binding. Exchange of three amino acid residues adjacent to the basic DNA contact regions between Fos and Jun reversed the binding orientation. The effects of these amino acid residues on the orientation of heterodimer binding depended on base-pairs flanking the core AP-1 recognition sequence. Single amino acid and base-pair substitutions had parallel effects on DNA bending by Fos-Jun-AP-1 complexes and on heterodimer orientation. The binding orientation exhibited a close correspondence with both the difference in bending propensities of opposite sides of the AP-1 site as well as the difference in bending potentials of the Fos and Jun subunits of the heterodimer. The influence of flanking DNA sequences on heterodimer orientation was attenuated in the presence of high concentrations of multivalent cations. Base substitutions up to one helical turn from the center of the AP-1 site affected the binding orientation. Modification of flanking base-pairs with positively or negatively charged functional groups had opposite effects on the orientation of heterodimer binding. These changes in DNA charge had converse effects on the orientation preferences of heterodimers in which charged amino acid residues adjacent to the basic regions were exchanged between Fos and Jun. These results indicate that the orientation of heterodimer binding is determined primarily by minimization of the electrostatic free energy of the Fos-Jun-AP-1 complex. Consequently, long-range electrostatic interactions influence the architecture of nucleoprotein complexes.

Amino Acid Sequence↗

PubMed1991

Molecular analysis of the responder satellite DNA in Drosophila melanogaster: DNA bending, nucleosome structure, and Rsp-binding proteins.

The Responder (Rsp) locus of Drosophila melanogaster, the target locus of segregation distortion, is a satellite DNA array. This repeat array imparts some fitness advantage to the chromosomes bearing it. In this paper, we report the following three related molecular properties of this satellite repeat: (1) Sequence-directed curvature--On a polyacrylamide gel, Rsp-containing fragments migrate slower than would be predicted on the basis of their physical sizes. The extent of migration retardation correlates with the size and position of the Rsp sequence in a DNA fragment, suggesting that Rsp DNA is bent. The bending is shown to be affected by a DNA-binding drug (Hoechst 33258). (2) Nucleosome structure--Nucleosomes associated with Rsp repeats have an unusual spacing pattern. Instead of being spaced at approximately 190-bp intervals as is the bulk chromatin, they are separated at approximately 240-bp intervals, roughly the size of a dimeric Rsp repeat. The nucleosomal structure in the Rsp region is preferentially disrupted by Hoechst 33258, whereas the bulk chromatin appears to be insensitive to the drug. (3) Rsp-DNA binding proteins--Gel mobility-shift assays using nuclear extracts from pupae and end-labeled Rsp repeat demonstrate the presence of three distinct DNA-protein complexes. Competition assays suggest that these complexes are specific to the Rsp sequence, and two of these nucleoprotein complexes seem to be influenced by the presence of Hoechst 33258. The observed complexes are formed by nonhistone proteins of somatic origin and may be related to the normal functions of Rsp, rather than to the germ-line segregation distortion activities.

Animals↗

PubMed1989

Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication.

Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.

Bacteriophage lambda↗

PubMed2004

Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence.

The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.

Carrier Proteins↗

PubMed1991

A possible role of the viral core protein in the regulation of assembly and disassembly of alphavirus cores.

In many viral systems the genome is part of a stable nucleoprotein complex which has to be dissociated early in infection, in order to make the genome accessible for transcription or translation. It is not directly evident how a nucleoprotein generated as a stable complex during virus assembly can be dissociated during the initial steps of viral multiplication. During analyses of the molecular biology of alphavirus replication it has been shown that the core protein of these viruses is a multifunctional protein. In this manuscript I propose that one of the functions of the core protein, namely its ability to bind to the large subunit of the cellular ribosomes both during synthesis and as a mature protein, allows to regulate the assembly and disassembly of alphavirus cores. I propose that in the early stages of virus infection the core protein is transferred from the core of the incoming virus to the cellular ribosomes, whereas in the late stages of infection when a significant part of the ribosomes contain bound, newly synthesized core protein an efficient transfer of newly synthesized core protein into cores can occur.

Alphavirus↗

PubMed2003

HMGA1 co-activates transcription in B cells through indirect association with DNA.

The immunoglobulin heavy chain enhancer, or mu enhancer, is required for B cell development. Only the appropriate combination of transcription factors results in B cell-specific enhancer activation. HMGA1 (formerly (HMG-I(Y)) is a proposed co-activator of the ETS transcription factors required for mu enhancer activity. HMGA1 associates with the ETS factor PU.1, resulting in changes in PU.1 structure, and enhanced transcriptional synergy with Ets-1 on the mu enhancer in nonlymphoid cells. New data show HMGA1 directly interacts with Ets-1 in addition to PU.1. In vitro HMGA1/Ets-1 interaction facilitates Ets-1/mu enhancer binding in the absence of an HMGA1.Ets-1.DNA complex. To address whether HMGA1 is present in the transcriptionally active mu nucleoprotein complex, we completed DNA pull-down assays to detect protein tethering in the context of protein/DNA interaction. Results show that HMGA1 is not tightly associated with mu enhancer DNA through PU.1 or Ets-1, despite strong associations between these proteins in solution. However, chromatin immunoprecipitation assays show HMGA1 associates with the endogenous enhancer in B cells. Furthermore, antisense HMGA1 substantially decreases mu enhancer activity in B cells. Taken together, these data suggest that HMGA1 functions as a transcriptional mu enhancer co-activator in B cells through indirect association with DNA.

Animals↗

PubMedDate not supplied

[Characteristics of protein-DNA association in chromatin from epidermal cells in psoriasis].

Two forms of nucleoprotein complex alpha and beta were found in human keratinocytes by means of nucleoprotein celite chromatography. They were not observed any principal differences between distribution of chromatin various forms in impaired and normal skin of the patients with psoriasis as well as in skin of healthy persons. Only slight differences were detected in the ratio of radioactive DNA in alpha and beta forms of chromatin from impaired and normal keratinocytes, while tightly-bound DNA was increased in psoriasis. In psoriasis acceleration of keratinocyte cell cycle but not an increase in proliferating cells pool appear to occur.

Cell Division↗

PubMed1977

Characterization of a soluble simian-virus-40 transcription complex.

We have previously described the isolation, from nuclei of monkey cells infected with Simian virus 40 (SV40), of a nucleoprotein complex which is able to achieve viral transcription. This complex contains SV40 DNA and RNA polymerase II molecules which have initiated transcription during the viral development. We show here, by molecular hybridization experiments, that most of the templates active in SV40 transcription can be dissociated from host DNA. In conditions where supercoiled SV40 DNA form I sediments at 21 S, the transcription complex has a sedimentation coefficient of about 25 S. Inhibition of viral DNA synthesis by cytosine arabinonucleoside or chloroquine does not affect the activity of the transcription complex, which suggests that replicating molecules are not required for viral RNA synthesis and that SV40 DNA form I could serve as template for late SV40 transcription. A large fraction of the RNA synthesized in vitro remains associated with the SV40 DNA template in cesium sulfate density gradient. The RNA chains produced by the complex are heterogeneous in size, most of them being as large or larger than the viral genome.

Cell Line↗

PubMed2005

Assembly of the mariner Mos1 synaptic complex.

The mobility of transposable elements via a cut-and-paste mechanism depends on the elaboration of a nucleoprotein complex known as the synaptic complex. We show here that the Mos1 synaptic complex consists of the two inverted terminal repeats of the element brought together by a transposase tetramer and is designated paired-end complex 2 (PEC2). The assembly of PEC2 requires the formation of a simpler complex, containing one terminal repeat and two transposase molecules and designated single-end complex 2 (SEC2). In light of the formation of SEC2 and PEC2, we demonstrate the presence of two binding sites for the transposase within a single terminal repeat. We have found that the sequence of the Mos1 inverted terminal repeats contains overlapping palindromic and mirror motifs, which could account for the binding of two transposase molecules "side by side" on the same inverted terminal repeat. We provide data indicating that the Mos1 transposase dimer is formed within a single terminal repeat through a cooperative pathway. Finally, the concept of a tetrameric synaptic complex may simply account for the inability of a single mariner transposase molecule to interact at the same time with two kinds of DNA: the inverted repeat and the target DNA.

Animals↗

PubMed2003

The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein.

Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.

Binding, Competitive↗

PubMed1995

Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy.

Gene activation frequently requires an array of proteins bound to sites distal to the transcription start site. The assembly of these protein-bound sites into specialized nucleoprotein complexes is a prerequisite for transcriptional activation. Structural analysis of these higher order complexes will provide crucial information for understanding the mechanisms of gene activation. We have used both electron microscopy and scanning force microscopy to elucidate the structure of complexes formed between DNA and heat-shock transcription factor (HSF) 2, a human heat-shock transcriptional activator that binds DNA as a trimer. Electron microscopy reveals that HSF2 will bring together distant DNA sites to create a loop. We show that this association requires only the DNA binding and trimerization domains of HSF2. Metal shadowing techniques used for electron microscopy obscure details of these nucleoprotein structures. Greatly increased resolution was achieved by directly imaging the complexes in the scanning force microscope, which reveals that at least two trimers are required for the association of HSF2-bound DNA sites.

DNA↗

PubMedDate not supplied

[Possible role of iron ions in changes in composition of DNA complexes and their magnetic properties in cell cycle processes].

A hypothesis on the possible role of iron cations in transformations of nucleoprotein complexes at certain stages of the cell cycle was formulated. The central idea of the hypothesis is the proposition that iron ions provide changes in the composition of complexes formed by phosphate groups of DNA, by substituting effectively the cationic amino groups of organic ligands in these complexes by the mechanism of competitive replacement of ligands. Then iron ions can be removed by changing the charge of iron ions bound to DNA during redox reactions and their transfer to mobile complexes or by the formation and subsequent removal of weakly charged magnetic nanoparticles of ferric oxides. These magnetic nanoparticles may be responsible for the magnetic effects in cells, e.g., broad ESR signals inherent in ferromagnetic systems. These magnetic effects were discovered in DNA preparations and in cell cultures at early stages of cell division by L.A. Blumenfeld and coworkers.

Cations↗

PubMed2002

The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome.

The mycelial prokaryote Streptomyces coelicolor A3(2) possesses a large linear chromosome (8.67 Mb) with a centrally located origin of replication (oriC). Recently, chromosome partitioning genes (parA and parB) and putative ParB binding sites (parS sequences) were identified in its genome. The S. coelicolor chromosome contains more parS sequences than any other bacterial chromosome characterized so far. Twenty of the 24 parS sequences are densely packed within a relatively short distance (approximately 200 kb) around oriC. A series of in vitro and in vivo experiments showed that S. coelicolor ParB protein interacts specifically with the parS sequences, albeit with a rather low affinity. Our results suggested that the binding of ParB is not only determined by the parS sequence, but also by the location of target DNA close to oriC. The unusually high number and close proximity to each other of the parS sites, together with in vivo and in vitro evidence that multiple ParB molecules may assemble along the DNA from an initial ParB-parS complex, suggest that a large DNA segment around the replication origin may form a massive nucleoprotein complex as part of the replication-partitioning cycle.

Bacterial Proteins↗

PubMed1988

Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes.

Using morphological and cell biological techniques, we have shown that the RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs. The results suggest that these cytopathic vacuoles constitute sites not only for viral RNA synthesis, but also for translation of structural proteins, and for the assembly of nucleocapsids.

Alphavirus↗

PubMed1998

Ku antigen binds to Alu family DNA.

The GC-rich segment containing GGAGGC (Alu core) is conserved within the RNA polymerase III (pol III) promoters of Alu family sequences. We have shown that the GGAGGC motif functions as a modulator of DNA replication as well as of transcription, and identified the proteins binding to the motif in human HeLa cells. In this study, the Alu core binding proteins were partially purified from human Raji cells by using an Alu core DNA affinity column. Both the proteins thus purified were implied to be subunits of Ku antigen based on the following criteria: The molecular weights of the proteins estimated on gel electrophoreses were 70 and 85 kDa, respectively, under denaturing conditions, while under non-denaturing conditions only one band was observed for the same sample at 150 kDa, probably representing hetero-dimer formed between the 70 and 85 kDa proteins. The sizes and the hetero-dimer formation are reminiscent of the 70 and 80 kDa subunits of Ku antigen (Ku-p70 and Ku-p80). Moreover, the purified proteins were immunoreactive with anti-Ku antibodies, and the specific DNA-protein complex on the Alu core element was cancelled by the anti-Ku antibodies. The nucleoprotein complex showed the same clipping patterns as those of the complex between the Alu core element and an authentically purified Ku antigen after proteolytic cleavage with trypsin and chymotrypsin.

Antigens, Nuclear↗