Stable Nucleoprotein Research Articles

Multivalent interactions essential for lentiviral integrase function

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits1. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.

The Ubiquitin Ligase TRAIP: Double-Edged Sword at the Replisome.

In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity.

Roles of the INO80 and SWR1 Chromatin Remodeling Complexes in Plants.

Eukaryotic genes are packed into a dynamic but stable nucleoprotein structure called chromatin. Chromatin-remodeling and modifying complexes generate a dynamic chromatin environment that ensures appropriate DNA processing and metabolism in various processes such as gene expression, as well as DNA replication, repair, and recombination. The INO80 and SWR1 chromatin remodeling complexes (INO80-c and SWR1-c) are ATP-dependent complexes that modulate the incorporation of the histone variant H2A.Z into nucleosomes, which is a critical step in eukaryotic gene regulation. Although SWR1-c has been identified in plants, plant INO80-c has not been successfully isolated and characterized. In this review, we will focus on the functions of the SWR1-c and putative INO80-c (SWR1/INO80-c) multi-subunits and multifunctional complexes in Arabidopsis thaliana. We will describe the subunit compositions of the SWR1/INO80-c and the recent findings from the standpoint of each subunit and discuss their involvement in regulating development and environmental responses in Arabidopsis.

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

Regulation of Plant Growth and Development: A Review From a Chromatin Remodeling Perspective.

In eukaryotes, genetic material is packaged into a dynamic but stable nucleoprotein structure called chromatin. Post-translational modification of chromatin domains affects the expression of underlying genes and subsequently the identity of cells by conveying epigenetic information from mother to daughter cells. SWI/SNF chromatin remodelers are ATP-dependent complexes that modulate core histone protein polypeptides, incorporate variant histone species and modify nucleotides in DNA strands within the nucleosome. The present review discusses the SWI/SNF chromatin remodeler family, its classification and recent advancements. We also address the involvement of SWI/SNF remodelers in regulating vital plant growth and development processes such as meristem establishment and maintenance, cell differentiation, organ initiation, flower morphogenesis and flowering time regulation. Moreover, the role of chromatin remodelers in key phytohormone signaling pathways is also reviewed. The information provided in this review may prompt further debate and investigations aimed at understanding plant-specific epigenetic regulation mediated by chromatin remodeling under continuously varying plant growth conditions and global climate change.

Nucleoprotein Intermediates in HIV-1 DNA Integration: Structure and Function of HIV-1 Intasomes.

Integration of a DNA copy of the viral genome into host DNA is an essential step in the replication cycle of HIV-1 and other retroviruses and is an important therapeutic target for drugs. DNA integration is catalyzed by the viral integrase protein and proceeds through a series of stable nucleoprotein complexes of integrase, viral DNA ends and target DNA. These nucleoprotein complexes are collectively called intasomes. Retroviral intasomes undergo a series of transitions between initial formation and catalysis of the DNA cutting and joining steps of DNA integration. Intasomes, rather than free integrase protein, are the target of currently approved drugs that target HIV-1 DNA integration. High-resolution structures of HIV-1 intasomes are needed to understand their detailed mechanism of action and how HIV-1 may escape by developing resistance. Here, we focus on our current knowledge of the structure and function of HIV-1 intasomes, with reference to related systems as required to put this knowledge in context.

CIITA: A Master Regulator of Adaptive Immunity Shows Its Innate Side in the Bone

CIITA (the Major Histocompatibility Class (MHC) II transactivator) has long been known as the master regulator of MHC Class II genes, which are critical for normal immune function (reviewed in (1, 2)). CIITA is a non-DNA binding co-activator that specifically regulates expression of Major Histocompatibility Class II molecules, with CIITA deficiency leading to the rare human immunodeficiency disease termed Bare Lymphocyte Syndrome (3). CIITA also regulates expression of genes encoding accessory proteins required for MHCII-restricted antigen presentation, thus CIITA is central to controlling the response to foreign antigens and the maintenance of tolerance (1, 2). In this issue of the Journal of Bone and Mineral Research, Benasciutti and colleagues demonstrate that CIITA is also a major regulator of osteoclast differentiation and bone homeostasis (4).

Determining the topology of stable protein–DNA complexes

Difference topology is an experimental technique that can be used to unveil the topological structure adopted by two or more DNA segments in a stable protein-DNA complex. Difference topology has also been used to detect intermediates in a reaction pathway and to investigate the role of DNA supercoiling. In the present article, we review difference topology as applied to the Mu transpososome. The tools discussed can be applied to any stable nucleoprotein complex.

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

DNA requirements for assembly and stability of HIV‐1 intasomes

Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV-1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long-nonspecific internal DNA for intasome assembly. Although intasomes are unstable in solution when the nonspecific internal DNA is cut off after assembly, they are stable in agarose gels. These complexes are indistinguishable from SSCs with nonspecific internal DNA in Förster resonance energy transfer (FRET) experiments suggesting the interactions with the viral DNA and integrase tetramer are the same regardless of the presence of nonspecific internal DNA. We discuss models of how the internal DNA contributes to intasome assembly and stability. FRET is exquisitely sensitive to the distance between the fluorophores and given certain assumptions can be translated to distance measurements. We anticipated that a set of such distance constraints would provide a map of the DNA path within the intasome. In reality, the constraints we could impose from the FRET data were quite weak allowing a wide envelope for the possible path. We discuss the difficulties of converting the FRET signal to absolute distance within nucleoprotein complexes.

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a approximately 1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21-24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20-50 nucleotides was approximately 3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.

Nucleoprotein complex intermediates in HIV-1 integration

Integration of retroviral DNA into the host genome is an essential step in the viral replication cycle. The viral DNA, made by reverse transcription in the cytoplasm, forms part of a large nucleoprotein complex called the preintegration complex (PIC). The viral integrase protein is the enzyme within the PIC that is responsible for integrating the viral DNA into the host genome. Integrase is tightly associated with the viral DNA within the PIC as demonstrated by functional assays. Integrase protein catalyzes the key DNA cutting and joining steps of integration in vitro with DNA substrates that mimic the ends of the viral DNA. Under most in vitro assay conditions the stringency of the reaction is relaxed; most products result from “half-site” integration in which only one viral DNA end is integrated into one strand of target DNA rather than concerted integration of pairs of DNA as occurs with PICs and in vivo. Under these relaxed conditions catalysis appears to occur without formation of the highly stable nucleoprotein complexes that is characteristic of the association of integrase with viral DNA in the PIC. Here we describe methods for the assembly of nucleoprotein complex intermediates in HIV-1 DNA integration from purified HIV-1 integrase and substrates that mimic the viral DNA ends.

Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences

Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop. Using single-molecule and bulk biochemical approaches, we found that Tn5 transposase assembles a stable nucleoprotein complex in the absence of Tn5 transposon end sequences. Surprisingly, this end sequence-independent complex has structural similarities to the synaptic complex. This complex is the node of a DNA loop; transposase dimerization and DNA specificity mutants affect its assembly; and it likely has the same number of proteins and DNA molecules as the synaptic complex. Furthermore, our results indicate that Tn5 transposase preferentially binds and loops a subset of non-Tn5 end sequences. Assembly of end sequence-independent nucleoprotein complexes likely plays a role in the in vivo downregulation of transposition and the cis-transposition bias of many bacterial transposases.

Retroviral DNA integration: reaction pathway and critical intermediates

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.

Dps protects cells against multiple stresses during stationary phase.

Dps, the nonspecific DNA-binding protein from starved cells, is the most abundant protein in stationary-phase Escherichia coli. Dps homologs are found throughout the bacteria and in at least one archaeal species. Dps has been shown to protect cells from oxidative stress during exponential-phase growth. During stationary phase, Dps organizes the chromosome into a highly ordered, stable nucleoprotein complex called the biocrystal. We show here that Dps is required for long-term stationary-phase viability under competitive conditions and that dps mutants have altered lag phases compared to wild-type cells. We also show that during stationary phase Dps protects the cell not only from oxidative stress but also from UV and gamma irradiation, iron and copper toxicity, thermal stress, and acid and base shock. The protective roles of Dps are most likely achieved through a combination of functions associated with the protein-DNA binding and chromosome compaction, metal chelation, ferroxidase activity, and regulation of gene expression.

Small, Highly Structured RNAs Participate in the Conversion of Human Recombinant PrP Sen to PrP Resin Vitro

We have identified a small, highly structured (shs)RNA that binds human recombinant prion protein (hrPrP) with high affinity and specificity under physiological conditions (e.g. 10% bovine calf serum (BCS), neutral pH, nanomolar concentrations of RNA and hrPrP). We also demonstrate the ability of this shsRNA to form highly stable nucleoprotein complexes with hrPrP and cellular PrP (PrP C) from various cell extracts and mammalian brain homogenates. The apparent mass of the nucleoprotein complex is dependent on the molar ratio of hrPrP to RNA during complex formation. The hrPrP in these complexes acquires resistance to degradation by Proteinase K (PK). Other shsRNAs, however, under identical conditions, neither form stable complexes with hrPrP nor do they induce resistance to PK digestion. We also demonstrate that the RNAs in these nucleoprotein complexes become resistant to ribonuclease A hydrolysis. These interactions between shsRNAs and hrPrP suggest possible roles of RNAs in the modulation of PrP structure and perhaps disease development. ShsRNAs that bind to hrPrP with high affinity and induce resistance to PK digestion can be used to develop molecular biology assays for the screening of compounds associated with PrP structure transformation or for drugs that inhibit this process.

CIITA and the MHCII Enhanceosome in the Regulation of MHCII Expression

Major Histocompatibility Complex class II (MHCII) molecules direct the development, activation and homeostasis of CD4+ T cells. Given these key functions it is not surprising that the absence of MHCII expression results in a severe primary immunodeficiency disease called MHCII deficiency or the Bare Lymphocyte Syndrome (BLS). The genetic defects responsible for BLS lie in genes encoding transcription factors required for MHCII expression. Four different MHCII regulatory genes encoding RFXANK, RFX5, RFXAP and CIITA have been identified. The first three are subunits of RFX, a ubiquitously expressed factor that binds cooperatively with other proteins to MHCII and related promoters to form a highly stable macromolecular nucleoprotein complex referred to as the MHCII enhanceosome. This enhanceosome serves as a landing pad for the MHCII transactivator CIITA. CIITA is a non-DNA binding coactivator that serves as the master control factor for MHCII expression. The highly regulated expression pattern of CIITA ultimately dictates the cell type specificity, induction and level of MHCII expression. The enhanceosome and CIITA collaborate in activating transcription by promoting histone hyperacetylation and by recruiting components of the general transcription machinery. In this review we summarize what is known about the molecular basis of BLS and what this has taught us about the mechanisms regulating transcription of MHCII and related genes. Particular attention is devoted to the structure, function and mode of action of the MHCII enhanceosome and CIITA. In addition, we focus on the highly regulated and cell type specific expression of CIITA.

Analysis of CREB mutants in Tax complex formation and trans-activation.

The human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax interacts with cellular transcription factors to facilitate viral replication in infected cells. Tax binds to the cellular transcription factor CREB and the cellular coactivator protein CBP to form a stable nucleoprotein complex on the viral enhancer elements. The formation of this complex is believed to promote strong Tax-dependent transcriptional activation of viral gene expression. In this study, we characterize a series of internal CREB deletion mutants with respect to Tax and CBP recruitment and transcriptional activation. We find that, although several of these mutants are unable to support ternary complex formation with Tax and the viral CRE DNA, they are fully competent for cooperation with Tax in CBP recruitment. Unexpectedly, CREB proteins that carry deletions in a carboxyterminal region of the KID domain, while competent for ternary and quaternary complex formation, were defective for Tax trans-activation in vivo. These studies suggest that CREB may serve more than just a "scaffolding" role in Tax trans-activation, cooperating directly with Tax (and CBP) to mediate strong transcriptional activation of the provirus.

RecA-mediated affinity capture: a method for full-length cDNA cloning.

We describe an improved method for rapid cloning of full-length cDNA from cDNA libraries. This approach is based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-stranded DNA probe and homologous sequences in circular double-stranded DNA. Hybridization of RecA-coated biotinylated DNA probes to homologous plasmid DNA creates triple-stranded complexes, which are then captured on streptavidin-coated magnetic beads. Following magnetic separation of the hybrid molecules, the enriched plasmid population is recovered by alkaline treatment, precipitated, resuspended and used to transform bacteria. Typically, many clones can then be recovered by colony hybridization screening of a single plate of the enriched library. We have used this technology to clone full-length and alternatively spliced forms of the human bcl-xL cDNA from a human liver cDNA library.

The role of HMG I(Y) in the assembly and function of the IFN-beta enhanceosome.

Transcriptional activation of the virus inducible enhancer of the human interferon-beta (IFN-beta) gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappaB, ATF-2/c-Jun, IRFs and the architectural protein of the mammalian high mobility group I(Y) [HMG I(Y)]. Here, we demonstrate that the first step in enhanceosome assembly, i.e. HMG I(Y)-dependent recruitment of NF-kappaB and ATF-2/c-Jun to the enhancer, is facilitated by discrete regions of HMG I and is mediated by allosteric changes induced in the DNA by HMG I(Y) and not by protein-protein interactions between HMG I(Y) and these proteins. However, we show that completion of the enhanceosome assembly process requires protein-protein interactions between HMG I(Y) and the activators. Finally, we demonstrate that once assembled, the IFN-beta enhanceosome is an unusually stable nucleoprotein structure that can activate transcription at high levels by promoting multiple rounds of reinitiation of transcription.