Ori Sequences Research Articles

Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis in Corynebacterium glutamicum.

Lycopene represents one of the central compounds in the carotenoid pathway and it exhibits a potent antioxidant ability with wide potential applications in medicine, food, and cosmetics. The microbial production of lycopene has received increasing concern in recent years. Corynebacterium glutamicum (C. glutamicum) is considered to be a safe and beneficial industrial production platform, naturally endowed with the ability to produce lycopene. However, the scarcity of efficient genetic tools and the challenge of identifying crucial metabolic genes impede further research on C. glutamicum for achieving high-yield lycopene production. To address these challenges, a novel genetic editing toolkit, CRISPR/MAD7 system, was established and developed. By optimizing the promoter, ORI and PAM sequences, the CRISPR/MAD7 system facilitated highly efficient gene deletion and exhibited a broad spectrum of PAM sites. Notably, 25 kb of DNA from the genome was successfully deleted. In addition, the CRISPR/MAD7 system was effectively utilized in the metabolic engineering of C. glutamicum, allowing for the simultaneous knockout of crtEb and crtR genes in one step to enhance the accumulation of lycopene by blocking the branching pathway. Through screening crucial genes such as crtE, crtB, crtI, idsA, idi, and cg0722, an optimal carotenogenic gene combination was obtained. Particularly, cg0722, a membrane protein gene, was found to play a vital role in lycopene production. Therefore, the CBIEbR strain was obtained by overexpressing cg0722, crtB, and crtI while strategically blocking the by-products of the lycopene pathway. As a result, the final engineered strain produced lycopene at 405.02 mg/L (9.52 mg/g dry cell weight, DCW) in fed-batch fermentation, representing the highest reported lycopene yield in C. glutamicum to date. In this study, a powerful and precise genetic tool was used to engineer C. glutamicum for lycopene production. Through the modifications between the host cell and the carotenogenic pathway, the lycopene yield was stepwise improved by 102-fold as compared to the starting strain. This study highlights the usefulness of the CRISPR/MAD7 toolbox, demonstrating its practical applications in the metabolic engineering of industrially robust C. glutamicum.

Alanine scan of WDV Rep reveals its ssDNA binding mechanism

HUH endonucleases (dubbed “HUH‐tags”) are small protein domains capable of forming covalent adducts with ssDNA in a sequence‐specific manner. The active site of HUH endonucleases utilizes a conserved histidine‐hydrophobic‐histidine motif to coordinate a metal ion that stabilizes a nucleophilic tyrosine necessary for phosphodiesterase activity. Because viral‐derived HUH‐tags are relatively small, react quickly, and require no chemical modifications to their ssDNA substrate, they have great value as protein fusion tags in biotechnologies ranging from genetic engineering to single‐molecule studies. One of the greatest assets of these tags is sequence‐specificity to their unique, native Ori sequence in vivo, introducing the possibility of using multiple HUH‐tags in multiplexed “one‐pot” reactions. However, their mechanism of ssDNA sequence binding and specificity is poorly understood, and there is noted cross‐reactivity between tags of closely‐related species. In order to understand the mechanism of ssDNA binding, we performed an alanine scan along a positively‐charged patch of one such HUH‐tag, wheat dwarf virus replication initiator protein (WDV), and characterized the enzymatic activity in both the rate and extent of the reaction. In molecular beacon Stopped‐Flow experiments, single point mutants of WDV showed a more than 60% decrease in reaction rate constant, and gel shift assays showed an almost complete lack of activity for some variants for single nucleotide ssDNA substitutions. In all, these findings help us to highlight key interactions in WDV‐ssDNA binding, and we gain further insight into potential rational engineering of HUH‐endonucleases to bind desired sequences of DNA.

The Functional 3D Organization of Unicellular Genomes

Genome conformation capture techniques permit a systematic investigation into the functional spatial organization of genomes, including functional aspects like assessing the co-localization of sets of genomic elements. For example, the co-localization of genes targeted by a transcription factor (TF) within a transcription factory. We quantify spatial co-localization using a rigorous statistical model that measures the enrichment of a subset of elements in neighbourhoods inferred from Hi-C data. We also control for co-localization that can be attributed to genomic order. We systematically apply our open-sourced framework, spatial-mHG, to search for spatial co-localization phenomena in multiple unicellular Hi-C datasets with corresponding genomic annotations. Our biological findings shed new light on the functional spatial organization of genomes, including: In C. crescentus, DNA replication genes reside in two genomic clusters that are spatially co-localized. Furthermore, these clusters contain similar gene copies and lay in genomic vicinity to the ori and ter sequences. In S. cerevisae, Ty5 retrotransposon family element spatially co-localize at a spatially adjacent subset of telomeres. In N. crassa, both Proteasome lid subcomplex genes and protein refolding genes jointly spatially co-localize at a shared location. An implementation of our algorithms is available online.

Mitochondrial Genomes of Flor Yeast Strains Are Characterized by Low Genetic Variability

High concentrations of ethanol and oxidative stress can cause a mutagenic effect on mitochondrial genomes (mtDNAs) of flor yeasts. We performed a comparative analysis of mtDNAs of wine yeast strains and three sequenced flor yeast strains. mtDNAs of flor strains are characterized by high conservation of nucleotide sequences and the structure. The numbers of destabilizing elements of mtDNA (GC clusters, short repeats, active ori sequences) in flor strains are generally lower than that in wine yeasts. The mtDNA conservation of flor strains can be determined by both bottleneck effects in the course of evolution and mechanisms aimed at maintaining the genetic stability. In general, the data obtained do not confirm the hypothesis of increased genetic variability of the mitochondrial genomes of flor yeast strains.

Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation.

ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA relA strain as a sensitized model to study factors affecting plasmid stability in the context of recombinant gene expression. We find that in this model, plasmid stability can be restored by two types of genetic modifications to the plasmid origin of replication (ori) sequence: point mutations and a novel 269nt duplication at the 5' end of the plasmid ori, which we named DAS (duplicated anti-sense) ori. Combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. In direct contradiction with the classic random distribution model, we find no correlation between increased plasmid copy number and increased plasmid stability. Increased stability cannot be explained by reduced levels of recombinant gene expression either. Our observations would be more compatible with a hybrid clustered and free-distribution model, which has been recently proposed based on detection of individual plasmids in vivo using super-resolution fluorescence microscopy. This work suggests a role for the plasmid ori in the control of segregation of ColE1 plasmids that is distinct from replication initiation, opening the door for the genetic regulation of plasmid stability as a strategy aimed at enhancing large-scale recombinant gene expression or bioremediation.

The fate of extrachromosomal DNAs in the progeny of plastid-transformed tobacco plants

Plastid transformation is conducted by homologous recombination. Plastid transformation using a vector-containing promoter and/or terminator sequences homologous to the plastid DNA for transgene expression often results in the generation of unintended extrachromosomal DNA molecules derived from the initial plastid DNA molecule via intramolecular recombination. All the extrachromosomal DNA molecules, including those lacking the ori sequence, found in the T0 plastid-transformed tobacco plants were still detected in the T3 plants. These results suggest that the extrachromosomal DNA molecules are newly generated from the initially transformed plastid DNA that is transmitted to the progeny during each generation. In addition, inward, outward, and overlap extension PCR of the extrachromosomal DNA molecules confirmed that these molecules were circular.

HsOrc4-Dependent Dna Remodeling of the ori-β Dhfr Replicator.

Replication of DNA in multicellular organisms initiates from origin of replication (ori) sequences, which significantly differ in length and complexity. One of the best characterized is hamster dihydrofolate reductase (DHFR), which contains the ori-β sequence with several functionally relevant domains, such as an AT-rich region, dinucleotide repeat element (DNR), sequence-induced bend DNA (BEND) and a RIP60 protein-binding site (RIP60). Prior to initiation, ori sequences are recognized by origin recognition complex (ORC), which is a hetero hexamer complex that serves as the landing pad for proteins of the pre-replication complex. The function of each ORC subunit is still unclear. In this study, we analyze the function of subunit 4 of the human ORC complex (HsOrc4) in interaction with a plasmid bearing the ori-β DHFR sequence. We show that the topologically closed DHFR ori-β replicator contains a bubble-like structure within its AT-rich region and that it is reversibly modified in the interaction with HsOrc4. The non-canonical structure of the AT-rich region in the topologically closed ori sequence is recognized and changed by HsOrc4 using the energy of supercoiled DNA. These findings could help to further elucidate DNA replication and its possible association with human genetic diseases.

Strand Invasion Structures in the Inverted Repeat of Candida albicans Mitochondrial DNA Reveal a Role for Homologous Recombination in Replication

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast.

CpG Islands: Starting Blocks for Replication and Transcription

ORI sequences. A few months ago, common features shared by large groups of ORIs were extracted from a large-scale mapping of replication origins based on the resistance of short nascent strands (SNS) to l-exonuclease digestion [5]. This study, conducted in HeLa cells and covering approximately 1% of the human genome, showed that origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no or only few origins in GC-poor regions. Moreover, half of the origins were mapped within or near CpG islands. In this issue of PLoS Genetics, a study by Sequeira-Mendes et al. extends this obser

PCR-based identification ofVibrio choleraeand the closely related speciesVibrio mimicususing the large chromosomalorisequence ofVibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.

Lessons from a small, dispensable genome: The mitochondrial genome of yeast

This article reviews the investigations on the mitochondrial genomes of yeast carried out in the author's laboratory during a quarter of a century (to be precise between 1966 and 1992). Our studies dealt with the structural basis for the cytoplasmic petite mutation, the replication, the transcription and the recombination of the mitochondrial genome, a genome which is dispensable and which comprises abundant non-coding sequences. This work led to some general conclusions on the nuclear genome of eukaryotes. Some recent results in apparent contradiction with our conclusions on ori sequences will also be briefly discussed.

Cryptic plasmid pRK2 from Escherichia coli W: sequence analysis and segregational stability

Cryptic plasmid pRK2 of the strain Escherichia coli W (ATCC 9637), an ancestor of production strains for penicillin G acylase, was sequenced and characterized. Based on the data on replication region and origin (ori sequence AAC, 924–926nt), the plasmid was classified as ColE1-like plasmid. DNA sequence analysis revealed five orfs hypothetical products of which shared a significant sequence similarity with putative proteins encoded by DNA of plasmid pColE1. orf1 codes for protein Rom involved in the control of plasmid replication, orfs 2–5 code for putative mobilization proteins (Mob A–D) that show a high level of similarity with the ones encoded by DNA of plasmids pColE1 and pLG13 (E. coli), pECL18 and pEC01 (Enterobacter cloacae), pSFD10 (Salmonella choleraesuis), and pScol7 (Shigella sonnei). Recombinant plasmids pRS11 (4.91kbp), pRS12 (4.91kbp), pRS2 (2.996kbp), and pRS3 (2.623kbp) that bear the Spectinomycin resistance determinant (SpcR) were prepared on the basis of nucleotide sequence of pRK2. These constructs are stably maintained in the population of E. coli cells grown in the absence of the selection pressure for 63 generations. The copy number of SpcR constructs in E. coli host grown in antibiotic-free LB medium ranges from 25 to 40 molecules per chromosomal equivalent.

Effects of sequences of prokaryotic origin on titer and transgene expression in retroviral vectors

Transcriptionally targeted MLV-based ProCon vectors allow expression of the transduced gene in a promoter-specific manner by replacement of the viral U3 region with a heterologous promoter. In order to evaluate the effects of sequence elements present in ProCon vectors on transgene expression (enhanced green fluorescence protein, EGFP), a series of deletion constructs mimicking the situation in proviral DNA following promoter conversion, where expression of the EGFP gene is driven by three different constitutive promoters (MLV U3, mCMV, and hCMV) in the context of a 5′LTR, respectively, were generated and tested in transient transfection experiments. We discovered that modifications in the 3′LTR have only marginal effects on the EGFP expression and the sequence between the promoter and the transgene did not influence EGFP expression at all. On the other hand, EGFP expression was reduced by up to 17-fold in cells transfected with constructs containing SV40neo and/or pBR322ori sequences. To study this effect in transduced cells, we generated a series of retroviral vectors in which these elements were deleted in various combinations and found that an increase in EGFP expression and viral titer was also consistently obtained using vectors lacking these elements, although this was much smaller than that observed using the expression constructs. A vector containing the gene for puromycin resistance (pac) in place of the neomycin resistance gene (neo) was also tested, and found to result in improved vector titers and transgene expression. We conclude that, where possible, the inclusion of neo and ori sequences in retroviral vectors should be avoided, and that, if selection of infected cells is necessary, the pac, rather than neo gene should be used.

In vivo excision and amplification of large human genomic segments using the Cre/loxP-and large T antigen/SV40 ori-mediated machinery

In vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a natural host, can be a powerful tool for obtaining the genomic sequences with minimum rearrangements. In this study, an in vivo excision and amplification system in human BJAB cells was devised by combining the Cre/loxP system of bacteriophage P1 and the large T antigen/SV40 ori system of Simian virus 40. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into 5′- and 3′-untranslated regions (UTRs) of the human iNOS. An SV40 ori sequence, which serves as a conditional replication system, was inserted between the loxP sites. Trans-acting genes cre and large T antigen, which were under the control of a tetracycline responsive promoter, were also inserted into the 5′- and 3′-UTRs of the iNOS, respectively, by homologous recombination. Upon induction by doxycycline, the 45-kb iNOS genomic fragment of human chromosome 17 flanked by two loxP sites was excised and amplified up to about 45 copies per cell. Our method is very useful for obtaining large genomic fragments in quantities directly from human cells without using foreign hosts. Therefore, our approach can be used effectively for gap sequencing of a genome, gene therapy, and functional analysis of unknown genes in human cells.

In vivo excision and amplification of large human genomic segments using the Cre/ loxP-and large T antigen/SV40 ori-mediated machinery

In vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a natural host, can be a powerful tool for obtaining the genomic sequences with minimum rearrangements. In this study, an in vivo excision and amplification system in human BJAB cells was devised by combining the Cre/ loxP system of bacteriophage P1 and the large T antigen/SV40 ori system of Simian virus 40. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into 5′- and 3′-untranslated regions (UTRs) of the human iNOS. An SV40 ori sequence, which serves as a conditional replication system, was inserted between the loxP sites. Trans-acting genes cre and large T antigen, which were under the control of a tetracycline responsive promoter, were also inserted into the 5′- and 3′-UTRs of the iNOS, respectively, by homologous recombination. Upon induction by doxycycline, the 45-kb iNOS genomic fragment of human chromosome 17 flanked by two loxP sites was excised and amplified up to about 45 copies per cell. Our method is very useful for obtaining large genomic fragments in quantities directly from human cells without using foreign hosts. Therefore, our approach can be used effectively for gap sequencing of a genome, gene therapy, and functional analysis of unknown genes in human cells.

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus.

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.

A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae.

The yeast mitochondrial chaperonin Hsp60 has previously been implicated in mitochondrial DNA (mtDNA) transactions: it is found in mtDNA nucleoids associated with single-stranded DNA; it binds preferentially to the template strand of active mtDNA ori sequences in vitro; and wild-type (ρ+) mtDNA is unstable in hsp60 temperature-sensitive (ts) mutants grown at the permissive temperature. Here we show that the mtDNA instability is caused by a defect in mtDNA transmission to daughter cells. Using high resolution, fluorescence deconvolution microscopy, we observe a striking alteration in the morphology of mtDNA nucleoids in ρ+ cells of an hsp60-t s mutant that suggests a defect in nucleoid division. We show that ρ− petite mtDNA consisting of active ori repeats is uniquely unstable in the hsp60-t s mutant. This instability of ori ρ− mtDNA requires transcription from the canonical promoter within the ori element. Our data suggest that the nucleoid dynamics underlying mtDNA transmission are regulated by the interaction between Hsp60 and mtDNA ori sequences.

Dual role of tumor suppressor p53 in regulation of DNA replication and oncogene e6-promoter activity of epidermodysplasia verruciformis-associated human papillomavirus type 8

Human papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3′ part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs “CCAAC” (nt 57–73) and M29 (nt 84–112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P 175. In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8.

The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus supports latent DNA replication in dividing cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is a multifunctional protein that is consistently expressed in all KSHV-associated malignancies. LANA interacts with a variety of cellular proteins, including the transcriptional cosuppressor complex mSin3 and the tumor suppressors p53 and Rb, thereby regulating viral and cellular gene expression. In addition, LANA is required for maintenance of the episomal viral DNA during latency in dividing cells. Colocalization studies suggest that LANA tethers the viral genome to chromosomes during mitosis. In support of this model, a specific LANA- binding site has recently been identified within the terminal repeat unit, and a chromatin interaction domain was mapped to a short amino acid stretch within the N-terminal domain of LANA. Epstein-Barr virus nuclear antigen 1 (EBNA-1), a functional homologue of LANA, is also required for genome segregation; in addition, EBNA-1 also supports efficient DNA replication of oriP-containing plasmids. By performing short-term replication assays, we demonstrate here for the first time that de novo synthesis of terminal-repeat (TR)-containing plasmids is highly dependent on the presence of LANA. We map the required cis-acting sequences within the TR to a 79-bp region and demonstrate that the DNA-binding domain of LANA is required for this DNA replication activity. Surprisingly, the 233-amino-acid C domain of LANA by itself partially supports replication. Our data show that LANA is a sequence-specific DNA-binding protein that, like EBNA-1, plays an important role in DNA replication and genome segregation. In addition, we show that all necessary cis elements for the origin of replication (ori) function are located within a single TR, suggesting that the putative ori of KSHV is different from those of other gammaherpesviruses, which all contain ori sequences within the unique long sequence outside of their TR. This notion is further strengthened by the unique modular structure of the KSHV TR element.

Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA.

Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA.