Rep Binding Site Research Articles

Estimating genetic diversity among selected wild grapevine genotypes from Southern Turkey by simple sequence repeat (SSR) and inter-Primer Binding Site(iPBS) markers

Estimating genetic diversity among selected wild grapevine genotypes from Southern Turkey by simple sequence repeat (SSR) and inter-Primer Binding Site(iPBS) markers

Widespread repression of anti-CRISPR production by anti-CRISPR-associated proteins.

Many bacteria use CRISPR-Cas systems to defend against invasive mobile genetic elements (MGEs). In response, MGEs have developed strategies to resist CRISPR-Cas, including the use of anti-CRISPR (Acr) proteins. Known acr genes may be followed in an operon by a putative regulatory Acr-associated gene (aca), suggesting the importance of regulation. Although ten families of helix-turn-helix (HTH) motif containing Aca proteins have been identified (Aca1-10), only three have been tested and shown to be transcriptional repressors of acr-aca expression. The AcrIIA1 protein (a Cas9 inhibitor) also contains a functionally similar HTH containing repressor domain. Here, we identified and analysed Aca and AcrIIA1 homologs across all bacterial genomes. Using HMM models we found aca-like genes are widely distributed in bacteria, both with and without known acr genes. The putative promoter regions of acr-aca operons were analysed and members of each family of bacterial Aca tested for regulatory function. For each Aca family, we predicted a conserved inverted repeat binding site within a core promoter. Promoters containing these sites directed reporter expression in E. coli and were repressed by the cognate Aca protein. These data demonstrate that acr repression by Aca proteins is widely conserved in nature.

Rapid Multilocus Adaptation of Clonal Cabbage Leaf Curl Virus Populations to Arabidopsis thaliana

Cabbage leaf curl virus (CabLCV) has a bipartite single-stranded DNA genome and infects the model plant Arabidopsis thaliana. CabLCV serves as a model for the genus Begomovirus, members of which cause tremendous crop losses worldwide. We have used CabLCV as a model for within-plant virus evolution by inoculating individual plants with infectious clones of either a wild-type or mutagenized version of the CabLCV genome. Consistent with previous reports, detrimental substitutions in the replication-associated ( Rep) gene were readily compensated for by direct reversion or alternative mutations. A surprising number of common mutations were detected elsewhere in both viral segments (DNA-A and DNA-B), indicating convergent evolution and suggesting that CabLCV may not be as well adapted to A. thaliana as commonly presumed. Consistent with this idea, a spontaneous coat protein variant consistently rose to high allele frequency in susceptible accession Columbia-0, at a higher rate than in hypersusceptible accession Sei-0. Numerous high-frequency mutations were also detected in a candidate Rep binding site in DNA-B. Our results reinforce the fact that spontaneous mutation of this type of virus occurs rapidly and can change the majority consensus sequence of a within-plant virus population in weeks. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Quaternary Structure and Deoxyribonucleic Acid-Binding Properties of the Heme-Dependent, CO-Sensing Transcriptional Regulator PxRcoM.

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, PxRcoM-1 requires all three repeat sites to cooperatively bind DNA with a [P]1/2 of 250 ± 10 nM and an average Hill coefficient, n, of 1.7 ± 0.1. The paralog PxRcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.

Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to in vitro evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy.

Rigidity enhances a magic-number effect in polymer phase separation

Cells possess non-membrane-bound bodies, many of which are now understood as phase-separated condensates. One class of such condensates is composed of two polymer species, where each consists of repeated binding sites that interact in a one-to-one fashion with the binding sites of the other polymer. Biologically-motivated modeling revealed that phase separation is suppressed by a “magic-number effect” which occurs if the two polymers can form fully-bonded small oligomers by virtue of the number of binding sites in one polymer being an integer multiple of the number of binding sites of the other. Here we use lattice-model simulations and analytical calculations to show that this magic-number effect can be greatly enhanced if one of the polymer species has a rigid shape that allows for multiple distinct bonding conformations. Moreover, if one species is rigid, the effect is robust over a much greater range of relative concentrations of the two species.

Molecular and pathogenic characterization of Cochliobolus anamorphs associated with common root rot of wheat in Azerbaijan

Genetic variation among the Azerbaijani isolates of anamorphs of Cochliobolus spp., the causal agents of common root rot of wheat, was evaluated using pathogenicity assessments, sequence analyses of the internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, as well as inter-simple sequence repeat (ISSR) and inter-primer binding site (iPBS) markers. Twenty-eight isolates used in this study were obtained from diseased wheat plants in cereal growing regions of Azerbaijan in 2017. Bipolaris sorokiniana, Curvularia spicifera, and Curvularia inaequalis were identified. Bipolaris sorokiniana isolates were the most virulent on wheat seedlings, followed by isolates of C. spicifera and C. inaequalis. Phylogenetic analyses of a combined dataset of the ITS and GPDH regions grouped the isolates into three clusters, each of which contained isolates of one species. The dendrogram derived from the unweighted pair-grouped method by arithmetic average (UPGMA) cluster analyses based on the data of ISSR and iPBS markers divided the isolates into three clusters in concordance to their taxonomic grouping at species level, but without correlation to their geographic origins. Population structure of isolates was estimated based on Bayesian modelling, and this showed three populations (K = 3) supporting the separation of isolates in the dendrogram with the greatest mean value of Ln likelihood (-893,8). Utilization of the markers either separately or together produced a high level of polymorphism at interspecies level, which allowed for the separation of species. Although both marker systems had similar discrimination power to reveal genetic differences among the species, ISSR markers were more informative for eliciting intraspecies polymorphisms within B. sorokiniana and C. spicifera isolates. This is the first study on genetic diversity and population structure of anamorphic stages of Cochliobolus associated with common root rot of wheat using iPBS markers.

CGGBP1 regulates CTCF occupancy at repeats

BackgroundCGGBP1 is a repeat-binding protein with diverse functions in the regulation of gene expression, cytosine methylation, repeat silencing and genomic integrity. CGGBP1 has also been identified as a cooperator of histone-modifying enzymes and as a component of CTCF-containing complexes that regulate the enhancer–promoter looping. CGGBP1–CTCF cross talk in chromatin regulation has been hitherto unknown.ResultsHere, we report that the occupancy of CTCF at repeats depends on CGGBP1. Using ChIP-sequencing for CTCF, we describe its occupancy at repetitive DNA. Our results show that endogenous level of CGGBP1 ensures CTCF occupancy preferentially on repeats over canonical CTCF motifs. By combining CTCF ChIP-sequencing results with ChIP sequencing for three different kinds of histone modifications (H3K4me3, H3K9me3 and H3K27me3), we show that the CGGBP1-dependent repeat-rich CTCF-binding sites regulate histone marks in flanking regions.ConclusionCGGBP1 affects the pattern of CTCF occupancy. Our results posit CGGBP1 as a regulator of CTCF and its binding sites in interspersed repeats.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

The Genomic Context and Corecruitment of SP1 Affect ERRα Coactivation by PGC-1α in Muscle Cells.

The peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) coordinates the transcriptional network response to promote an improved endurance capacity in skeletal muscle, eg, by coactivating the estrogen-related receptor-α (ERRα) in the regulation of oxidative substrate metabolism. Despite a close functional relationship, the interaction between these 2 proteins has not been studied on a genomic level. We now mapped the genome-wide binding of ERRα to DNA in a skeletal muscle cell line with elevated PGC-1α and linked the DNA recruitment to global PGC-1α target gene regulation. We found that, surprisingly, ERRα coactivation by PGC-1α is only observed in the minority of all PGC-1α recruitment sites. Nevertheless, a majority of PGC-1α target gene expression is dependent on ERRα. Intriguingly, the interaction between these 2 proteins is controlled by the genomic context of response elements, in particular the relative GC and CpG content, monomeric and dimeric repeat-binding site configuration for ERRα, and adjacent recruitment of the transcription factor specificity protein 1. These findings thus not only reveal a novel insight into the regulatory network underlying muscle cell plasticity but also strongly link the genomic context of DNA-response elements to control transcription factor-coregulator interactions.

Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome.

Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.

Presence of a trs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration.

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

Cloning and characterization of gene-resistant analogs (RGAs) involved in rust (Puccinia psidii) resistance in Eucalyptus grandis

Disease-resistant genes play an important role in defending against a variety of pathogens and insect pests in plants. Most of the disease-resistant genes encode proteins with conserved leucine rich repeat and nucleotide binding site domains. In this study, we cloned and characterized gene-resistant analogs (RGAs) from Eucalyptus grandis using degenerate PCR, with primers specifically targeting these two domains. The amplified fragments were cloned into the pGEM-T vector and transformed into Escherichia coli. Among the 90 clones obtained, 13 were sequenced and compared with each other and with previously identified gene-resistant diseases. A BLASTX search in GenBank revealed high similarities among the conserved domains of these cloned genes with RGA genes. Some clones, however, showed no significant similarity with DNA sequences in GenBank. Southern blotting analysis identified several polymorphic RFLP loci between distinct genotypes. However, none of them co-segregated with the Puccinia psidii Winter resistance gene 1 (Ppr1) in a population study.

95. Comprehensive wtAAV Integration Site Analysis Reveals Integration Site Formation Throughout the Whole Viral Genome

To establish latency, wildtype adeno-associated virus (wtAAV) is able to integrate its DNA into a specific region on chromosome 19 of the human genome, named AAVS1. This targeted integration mechanism relies on the function of the two larger versions (Rep78/68) of the Rep protein family, expressed by wtAAV. Rep is able to bind a 12 bp long sequence, called Rep binding site (RBS). The RBS can be found both in the viral ITRs and in AAVS1. By the formation of a trimeric complex between wtAAV, Rep and the genomic locus, targeted integration can be achieved. However, the exact mechanisms of Rep-mediated integration still remain unknown. To analyze wtAAV IS distribution genome-wide, large-scale integration analyses using high-throughput next generation sequencing have been performed. These studies revealed that Rep-mediated wtAAV integration preferentially occurs in AAVS1, but that additional integration hotspots associated with RBS can be found as well throughout the genome. Up to this point most integration sites (IS) reported for wtAAV were derived from vector-genome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most currently available methods for the analysis of viral IS rely on amplification of the vector-genome junction with primers that bind in the vicinity of the viral ends. Therefore, knowledge about the nature and structure of the internal wtAAV genome remains limited. To address this question, we investigated ~3e6 wtAAV concatemeric structures generated by IS analyses using linear amplification-mediated (LAM) PCR to identify potential alternative breakpoints throughout the wtAAV genome. We discovered that breakpoints occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter, there are a number of other preferred breakpoints throughout the wtAAV genome. Using a novel approach involving targeted enrichment of AAV and AAVS1 sequences, we also were able to consider viral IS at any position of the AAV genome. Targeted HiSeq sequencing of wtAAV and AAVS1 determined several hundreds of exact IS and confirmed that wtAAV genome junctions form at any position of its genome. Those cryptic integration events, not picked up by the majority of methods currently available, shed new light onto wtAAV persistence and may further improve current AAV persistence analyses in gene therapy.

Adeno-associated virus type 2 wild-type and vector-mediated genomic integration profiles of human diploid fibroblasts analyzed by third-generation PacBio DNA sequencing.

Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy.

Sequence-specific interactions of Rep proteins with ssDNA in the AT-rich region of the plasmid replication origin.

The DNA unwinding element (DUE) is a sequence rich in adenine and thymine residues present within the origin region of both prokaryotic and eukaryotic replicons. Recently, it has been shown that this is the site where bacterial DnaA proteins, the chromosomal replication initiators, form a specific nucleoprotein filament. DnaA proteins contain a DNA binding domain (DBD) and belong to the family of origin binding proteins (OBPs). To date there has been no data on whether OBPs structurally different from DnaA can form nucleoprotein complexes within the DUE. In this work we demonstrate that plasmid Rep proteins, composed of two Winged Helix domains, distinct from the DBD, specifically bind to one of the strands of ssDNA within the DUE. We observed nucleoprotein complexes formed by these Rep proteins, involving both dsDNA containing the Rep-binding sites (iterons) and the strand-specific ssDNA of the DUE. Formation of these complexes required the presence of all repeated sequence elements located within the DUE. Any changes in these repeated sequences resulted in the disturbance in Rep-ssDNA DUE complex formation and the lack of origin replication activity in vivo or in vitro.

A novel begomovirus isolated from sida contains putative cis- and trans-acting replication specificity determinants that have evolved independently in several geographical lineages.

A novel begomovirus isolated from a Sida rhombifolia plant collected in Sinaloa, Mexico, was characterized. The genomic components of sida mosaic Sinaloa virus (SiMSinV) shared highest sequence identity with DNA-A and DNA-B components of chino del tomate virus (CdTV), suggesting a vertical evolutionary relationship between these viruses. However, recombination analysis indicated that a short segment of SiMSinV DNA-A encompassing the plus-strand replication origin and the 5´-proximal 43 codons of the Rep gene was derived from tomato mottle Taino virus (ToMoTV). Accordingly, the putative cis- and trans-acting replication specificity determinants of SiMSinV were identical to those of ToMoTV but differed from those of CdTV. Modeling of the SiMSinV and CdTV Rep proteins revealed significant differences in the region comprising the small β1/β5 sheet element, where five putative DNA-binding specificity determinants (SPDs) of Rep (i.e., amino acid residues 5, 8, 10, 69 and 71) were previously identified. Computer-assisted searches of public databases led to identification of 33 begomoviruses from three continents encoding proteins with SPDs identical to those of the Rep encoded by SiMSinV. Sequence analysis of the replication origins demonstrated that all 33 begomoviruses harbor potential Rep-binding sites identical to those of SiMSinV. These data support the hypothesis that the Rep β1/β5 sheet region determines specificity of this protein for DNA replication origin sequences.

An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane

Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology.

The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria

Gene expression in plant mitochondria involves a complex collaboration of transcription initiation and termination, as well as subsequent mRNA processing to produce mature mRNAs. In this study, we describe the function of the Arabidopsis mitochondrial stability factor 1 (MTSF1) gene and show that it encodes a pentatricopeptide repeat protein essential for the 3′-processing of mitochondrial nad4 mRNA and its stability. The nad4 mRNA is highly destabilized in Arabidopsis mtsf1 mutant plants, which consequently accumulates low amounts of a truncated form of respiratory complex I. Biochemical and genetic analyses demonstrated that MTSF1 binds with high affinity to the last 20 nucleotides of nad4 mRNA. Our data support a model for MTSF1 functioning in which its association with the last nucleotides of the nad4 3′ untranslated region stabilizes nad4 mRNA. Additionally, strict conservation of the MTSF1-binding sites strongly suggests that the protective function of MTSF1 on nad4 mRNA is conserved in dicots. These results demonstrate that the mRNA stabilization process initially identified in plastids, whereby proteins bound to RNA extremities constitute barriers to exoribonuclease progression occur in plant mitochondria to protect and concomitantly define the 3′ end of mature mitochondrial mRNAs. Our study also reveals that short RNA molecules corresponding to pentatricopeptide repeat-binding sites accumulate also in plant mitochondria.

Inhibition of Binding of Tomato Yellow Leaf Curl Virus Rep to its Replication Origin by Artificial Zinc-Finger Protein

Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.