Structural analysis of a replication protein encoded by a plasmid isolated from a multiple sclerosis patient.
ABSTRACT: Bovine meat and milk factors (BMMFs) are circular, single-stranded episomal DNAs that have been detected in bovine meat and milk products. BMMFs are thought to have roles in human malignant and degenerative diseases. BMMFs encode a replication initiator protein (Rep) that is actively transcribed and translated in human cells. In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53?Å resolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five ?-helices, five ?-strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins.
Project description:The consumption of bovine milk and meat is considered a risk factor for colon- and breast cancer formation, and milk consumption has also been implicated in an increased risk for developing Multiple Sclerosis (MS). A number of highly related virus-like DNAs have been recently isolated from bovine milk and sera and from a brain sample of a MS patient. As a genetic activity of these Acinetobacter-related bovine milk and meat factors (BMMFs) is unknown in eukaryotes, we analyzed their expression and replication potential in human HEK293TT cells. While all analyzed BMMFs show transcriptional activity, the MS brain isolate MSBI1.176, sharing homology with a transmissible spongiform encephalopathy-associated DNA molecule, is transcribed at highest levels. We show expression of a replication-associated protein (Rep), which is highly conserved among all BMMFs, and serological tests indicate a human anti-Rep immune response. While the cow milk isolate CMI1.252 is replication-competent in HEK293TT cells, replication of MSBI1.176 is complemented by CMI1.252, pointing at an interplay during the establishment of persistence in human cells. Transcriptome profiling upon BMMF expression identified host cellular gene expression changes related to cell cycle progression and cell viability control, indicating potential pathways for a pathogenic involvement of BMMFs.
Project description:BACKGROUND: Repetitive extragenic palindromic elements (REPs) constitute a group of bacterial genomic repeats known for their high abundance and several roles in host cells´ physiology. We analyzed the phylogenetic distribution of particular REP classes in genomic sequences of sixty-three bacterial strains belonging to the Pseudomonas fluorescens species complex and ten strains of Stenotrophomonas sp., in order to assess intraspecific REP diversity and to gain insight into long-term REP evolution. RESULTS: Based on proximity to RAYT (REP-associated tyrosine transposase) genes, twenty-two and thirteen unique REP classes were determined in fluorescent pseudomonads and stenotrophomonads, respectively. In stenotrophomonads, REP elements were typically found in tens or a few hundred copies per genome. REPs of fluorescent pseudomonads were generally more numerous, occurring in hundreds or even over a thousand perfect copies of particular REP class per genome. REP sequences showed highly heterogeneous distribution. The abundances of REP classes roughly followed host strains´ phylogeny, differing markedly among individual clades. High abundances of particular REP classes appeared to depend on the presence of the cognate RAYT gene, and deviations from this state could be attributed to recent or ancient mutations of rayt-flanking REPs, or RAYT loss. RAYTs of both studied bacterial groups are monophyletic, and their cognate REPs show species-specific characteristics, suggesting shared evolutionary history of REPs, RAYTs and their hosts. CONCLUSIONS: The results of our large-scale analysis show that REP elements constitute intriguingly dynamic components of genomes of fluorescent pseudomonads and stenotrophomonads, and indicate that REP diversification and proliferation are ongoing processes. High numbers of REPs have apparently been retained during the entire evolutionary time since the establishment of these two bacterial lineages, probably because of their beneficial effect on host long-term fitness. REP elements in these bacteria represent a suitable platform to study the interplay between repeated elements, their mobilizers and host bacterial cells.
Project description:In bacterial plasmids, Rep proteins initiate DNA replication by undergoing a structural transformation coupled to dimer dissociation. Amyloidogenesis of the 'winged-helix' N-terminal domain of RepA (WH1) is triggered in vitro upon binding to plasmid-specific DNA sequences, and occurs at the bacterial nucleoid in vivo. Amyloid fibers are made of distorted RepA-WH1 monomers that assemble as single or double intertwined tubular protofilaments. RepA-WH1 causes in E. coli an amyloid proteinopathy, which is transmissible from mother to daughter cells, but not infectious, and enables conformational imprinting in vitro and in vivo; i.e. RepA-WH1 is a 'prionoid'. Microfluidics allow the assessment of the intracellular dynamics of RepA-WH1: bacterial lineages maintain two types (strains-like) of RepA-WH1 amyloids, either multiple compact cytotoxic particles or a single aggregate with the appearance of a fluidized hydrogel that it is mildly detrimental to growth. The Hsp70 chaperone DnaK governs the phase transition between both types of RepA-WH1 aggregates in vivo, thus modulating the vertical propagation of the prionoid. Engineering chimeras between the Sup35p/[PSI(+)] prion and RepA-WH1 generates [REP-PSI(+)], a synthetic prion exhibiting strong and weak phenotypic variants in yeast. These recent findings on a synthetic, self-contained bacterial prionoid illuminate central issues of protein amyloidogenesis.
Project description:REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpA(IS200/IS605)). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpA(REP)) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpA(REP) is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpA(REP) was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpA(REP) activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpA(REP) shared the same general organization and similar catalytic characteristics with TnpA(IS200/IS605) transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpA(REP) may therefore be one of the first examples of transposase domestication in prokaryotes.
Project description:Numerous metagenomic studies have uncovered a remarkable diversity of circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses, the majority of which are uncultured and unclassified. Unlike capsid proteins, the Reps show significant similarity across different groups of CRESS DNA viruses and have conserved domain organization with the N-terminal nuclease and the C-terminal helicase domain. Consequently, Rep is widely used as a marker for identification, classification and assessment of the diversity of CRESS DNA viruses. However, it has been shown that in certain viruses the Rep nuclease and helicase domains display incongruent evolutionary histories. Here, we systematically evaluated the co-evolutionary patterns of the two Rep domains across classified and unclassified CRESS DNA viruses. Our analysis indicates that the Reps encoded by members of the families Bacilladnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Nanoviridae and Smacoviridae display largely congruent evolutionary patterns in the two domains. By contrast, among the unclassified CRESS DNA viruses, 71% appear to have chimeric Reps. Such massive chimerism suggests that unclassified CRESS DNA viruses represent a dynamic population in which exchange of gene fragments encoding the nuclease and helicase domains is extremely common. Furthermore, purging of the chimeric sequences uncovered six monophyletic Rep groups that may represent new families of CRESS DNA viruses.
Project description:An approximately 4-Mb genomic segment on chromosome 17p11.2, commonly deleted in patients with the Smith-Magenis syndrome (SMS) and duplicated in patients with dup(17)(p11.2p11.2) syndrome, is flanked by large, complex low-copy repeats (LCRs), termed proximal and distal SMS-REP. A third copy, the middle SMS-REP, is located between them. SMS-REPs are believed to mediate nonallelic homologous recombination, resulting in both SMS deletions and reciprocal duplications. To delineate the genomic structure and evolutionary origin of SMS-REPs, we constructed a bacterial artificial chromosome/P1 artificial chromosome contig spanning the entire SMS region, including the SMS-REPs, determined its genomic sequence, and used fluorescence in situ hybridization to study the evolution of SMS-REP in several primate species. Our analysis shows that both the proximal SMS-REP (approximately 256 kb) and the distal copy (approximately 176 kb) are located in the same orientation and derived from a progenitor copy, whereas the middle SMS-REP (approximately 241 kb) is inverted and appears to have been derived from the proximal copy. The SMS-REP LCRs are highly homologous (>98%) and contain at least 14 genes/pseudogenes each. SMS-REPs are not present in mice and were duplicated after the divergence of New World monkeys from pre-monkeys approximately 40-65 million years ago. Our findings potentially explain why the vast majority of SMS deletions and dup(17)(p11.2p11.2) occur at proximal and distal SMS-REPs and further support previous observations that higher-order genomic architecture involving LCRs arose recently during primate speciation and may predispose the human genome to both meiotic and mitotic rearrangements.
Project description:Reactive energetic plasticizers (REPs) coupled with hydroxy-telechelic poly(glycidyl azide-co-tetrahydrofuran) (PGT)-based energetic polyurethane (PU) binders for use in solid propellants and plastic-bonded explosives (PBXs) were investigated. The generation of gem-dinitro REPs along with a terminal alkyne stemmed from a series of finely designed approaches to not only satisfy common demands as conventional energetic plasticizers, but also to prevent the migration of plasticizers. The miscibility and rheological behavior of a binary mixture of PGT/REP with various REP fractions were quantitatively determined by differential scanning calorimetry (DSC) and rheometer, respectively, highlighting the promising performance of REPs in the formulation process. The kinetics on the distinct reactivity of propargyl vs. 3-butynyl species of REPs towards the azide group of the PGT prepolymer in terms of Cu-free azide-alkyne 1,3-dipolar cycloaddition (1,3-DPCA) was studied by monitoring ¹H nuclear magnetic resonance spectroscopy and analyzing the activation energies (Ea) obtained using DSC. The thermal stability of the finally cured energetic binders with the incorporation of REPs indicated that the thermal stability of the REP/PGT-based PUs was maintained independently of the REP content. The tensile strength and modulus of the PUs increased with an increase in the REP content. In addition, the energetic performance and sensitivity of REP and REP triazole species was predicted.
Project description:Extragenic sequences in genomes, such as microRNA and CRISPR, are vital players in the cell. Repetitive extragenic palindromic sequences (REPs) are a class of extragenic sequences, which form nucleotide stem-loop structures. REPs are found in many bacterial species at a high copy number and are important in regulation of certain bacterial functions, such as Integration Host Factor recruitment and mRNA turnover. Although a new clade of putative transposases (RAYTs or TnpA(REP)) is often associated with an increase in these repeats, it is not clear how these proteins might have directed amplification of REPs. We report here the structure to 2.6?Å of TnpA(REP) from Escherichia coli MG1655 bound to a REP. Sequence analysis showed that TnpA(REP) is highly related to the IS200/IS605 family, but in contrast to IS200/IS605 transposases, TnpA(REP) is a monomer, is auto-inhibited and is active only in manganese. These features suggest that, relative to IS200/IS605 transposases, it has evolved a different mechanism for the movement of discrete segments of DNA and has been severely down-regulated, perhaps to prevent REPs from sweeping through genomes.
Project description:A family of artificial proteins, named ?Rep, based on a natural family of helical repeat was previously designed. ?Rep members are efficiently expressed, folded and extremely stable proteins. A large ?Rep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the ?Rep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display ?Rep binders with nanomolar dissociation constants against the GFP. The structures of two independent ?Rep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected ?Reps for GFP proved sufficient for practically useful applications such as pull-down experiments. ?Reps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific ?Reps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that ?Rep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes.
Project description:The families of viruses possessing single-stranded (ss) circular genome employ a dedicated replication initiator protein (Rep) for making copies of their genome through the process of rolling circle replication. The replication begins at conserved nonanucleotide sequence at the intergenic region. The Rep protein seems to be the most conserved amongst the available proteins of the nanovirids and comprises of the N-terminal endonuclease domain and the C-terminal helicase domain. The structural studies of Faba bean necrotic yellows virus endonuclease domain suggests a ??+?? fold comprising of central ? sheet built from five antiparallel ? strands surrounded by outer short ? helices. The catalysis is mediated by a conserved Tyr residue and employs divalent metal ions (Mn2+). On one hand, the Reps associate with each other and oligomerize and on the other hand interact with varied host and vector associated proteins for successful infection. The sequence analysis of Reps from previously known nanovirids and the newly found ones from metagenomics data shed light on the evolutionary pattern of nanovirids in comparison to other plant infecting ssDNA viruses.