Plasmid Origin Of Replication Research Articles

High Nucleotide Skew Palindromic DNA Sequences Function as Potential Replication Origins due to their Unzipping Propensity.

Locations of DNA replication initiation in prokaryotes, called "origins of replication", are well-characterized. However, a mechanistic understanding of the sequence dependence of the local unzipping of double-stranded DNA, the first step towards replication initiation, is lacking. Here, utilizing a Markov chain model that was created to address the directional nature of DNA unzipping and replication, we model the sequence dependence of local melting of double-stranded linear DNA segments. We show that generalized palindromic sequences with high nucleotide skews have a low kinetic barrier for local melting near melting temperatures. This allows for such sequences to function as potential replication origins. We support our claim with evidence for high-skew palindromic sequences within the replication origins of mitochondrial DNA, bacteria, archaea and plasmids.

Toward an understanding of the DNA replication initiation in bacteria.

Although the mechanism of DNA replication initiation has been investigated for over 50 years, many important discoveries have been made related to this process in recent years. In this mini-review, we discuss the current state of knowledge concerning the structure of the origin region in bacterial chromosomes and plasmids, recently discovered motifs recognized by replication initiator proteins, and proposed in the literature models describing initial origin opening. We review structures of nucleoprotein complexes formed by replication initiators at chromosomal and plasmid replication origins and discuss their functional implications. We also discuss future research challenges in this field.

K fragment as a polymerase chain reaction-based vector for antibiotic resistance gene hunting

Antimicrobial resistance (AMR) has emerged as an escalating health issue in the global public health arena. To evaluate and predict AMR, it is of utmost importance to identify and characterize both the known and unknown genes responsible for AMR. While known genes can be readily detected, identifying unknown genes present a challenge. In this study, we developed the plasmid K fragment (pKF) by modifying the pUC19 vector, specifically by removing the multiple cloning site and introducing a Prom-RBS sequence. pKF was used for amplification of K fragment that contains a ribosomal binding site (RBS), and promoter at both ends and plasmid origin of replication. The functionality of added Prom-RBS sequence and K fragment as a cloning vector was tested by cloning chloramphenicol resistance gene amplicon and erythromycin resistance gene from genomic DNA, respectively. The cloning experiment demonstrated the usability of this newly developed cloning method with K fragment. K fragment is an innovative vector that can be easily obtained through amplification by polymerase chain reaction and lacks antibiotic resistance markers. This novel approach is convenient to use since it allows cloning of resistance genes at all orientations and this flexibility can be maneuvered by changing restriction enzymes for primers and fragments. With these distinctive features, this vector stands out to be a versatile tool for cloning both known and unknown resistance genes, and the improved method with K fragment enables the microbiological and molecular characterization of cloned genes. K fragment can be utilized for cloning of resistance genes in bacteria originated in different environments without having to perform bacterial culture. We believe that the convenience brought by this technique could lend itself efficient in the battle against the growing AMR crisis through pre-emptive identification of resistance genes.

An Introduced RNA-Only Approach for Plasmid Curing via the CRISPR-Cpf1 System in Saccharomyces cerevisiae.

The CRISPR-Cas system has been widely used for genome editing due to its convenience, simplicity and flexibility. Using a plasmid-carrying Cas protein and crRNA or sgRNA expression cassettes is an efficient strategy in the CRISPR-Cas genome editing system. However, the plasmid remains in the cells after genome editing. Development of general plasmid-curing strategies is necessary. Based on our previous CRISPR-Cpf1 genome-editing system in Saccharomyces cerevisiae, the crRNA, designed for the replication origin of the CRISPR-Cpf1 plasmid, and the ssDNA, as a template for homologous recombination, were introduced for plasmid curing. The efficiency of the plasmid curing was 96 ± 4%. In addition, we further simplified the plasmid curing system by transforming only one crRNA into S. cerevisiae, and the curing efficiency was about 70%. In summary, we have developed a CRISPR-mediated plasmid-curing system. The RNA-only plasmid curing system is fast and easy. This plasmid curing strategy can be applied in broad hosts by designing crRNA specific for the replication origin of the plasmid. The plasmid curing system via CRISPR-Cas editing technology can be applied to produce traceless products without foreign genes and to perform iterative processes in multiple rounds of genome editing.

Emergence and Inter- and Intrahost Evolution of Pandrug-Resistant Klebsiella pneumoniae Coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2.

Klebsiella pneumoniae is capable of acquiring various exogenous genetic elements and subsequently conferring high antimicrobial resistance. Recently, a plasmid-mediated RND family multidrug efflux pump gene cluster, tmexCD1-toprJ1, was discovered in K. pneumoniae. In this study, we analyzed tigecycline-resistant K. pneumoniae isolates from patients from surveillance from 2017 to 2021. In addition to phenotype detection, including growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival, by whole-genome sequencing, we analyzed the phylogenetic relationships of the isolates harboring tmexCD1-toprJ1 and discovered the composition of plasmids carrying tmexCD1-toprJ1. In total, we discovered that 12 tigecycline-resistant isolates from 5 patients possessed tmexCD1-toprJ1, designated sequence type 22 (ST22) and ST3691. An ST11 isolate harbored a partial tmexD1, and a complete toprJ1 (tmexC1 was lost) was tigecycline sensitive. All the ST22 tigecycline-resistant isolates coharbored tmexCD1-toprJ1, blaNDM-1, and blaKPC-2. tmexCD1-toprJ1 was encoded by a novel IncU plasmid in ST22 and an IncFIB/HI1B plasmid in ST3691, which presented differences in mobility and stability. Interestingly, isolates from the same patients presented heteroresistance to tigecycline, not only among isolates from different specimens but also those from the same sample, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. Here, we reported the emergence of K. pneumoniae isolates coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2. The results highlight the impact of in vivo genetic heterogeneity of tmexCD1-toprJ1-carrying elements on the in vivo variation of tigecycline resistance, which might have notable influences on antimicrobial treatment. IMPORTANCE Pandrug-resistant (PDR) Klebsiella pneumoniae poses a great challenge to public health, and tigecycline is an essential choice for antimicrobial treatment. In this study, we reported the emergence of PDR K. pneumoniae coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2, which belongs to ST22 and ST3691. By whole-genome analysis, we reconstructed the evolutionary map of the ST22 ancestor to become the PDR superbug by acquiring multiple genetic elements encoding tmexCD1-toprJ1 or blaNDM-1. Importantly, the genetic contexts of tmexCD1-toprJ1 among the ST22 isolates are different and present with various mobilities and stabilities. Furthermore, we also discovered the heterogeneity of tigecycline resistance during long-term infection of ST22, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. This study tracks the inter- and intrahost microevolution of the superbug PDR K. pneumoniae and highlights the importance of timely monitoring of the variation of pathogens during antimicrobial treatment.

Cryo-EM Structure and Functional Studies of EBNA1 Binding to the Family of Repeats and Dyad Symmetry Elements of Epstein-Barr Virus oriP.

Epstein-Barr nuclear antigen 1 (EBNA1) is a multifunctional viral-encoded DNA-binding protein essential for Epstein-Barr virus (EBV) DNA replication and episome maintenance. EBNA1 binds to two functionally distinct elements at the viral origin of plasmid replication (oriP), termed the dyad symmetry (DS) element, required for replication initiation and the family of repeats (FR) required for episome maintenance. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the EBNA1 DNA binding domain (DBD) from amino acids (aa) 459 to 614 and its interaction with two tandem sites at the DS and FR. We found that EBNA1 induces a strong DNA bending angle in the DS, while the FR is more linear. The N-terminal arm of the DBD (aa 444 to 468) makes extensive contact with DNA as it wraps around the minor groove, with some conformational variation among EBNA1 monomers. Mutation of variable-contact residues K460 and K461 had only minor effects on DNA binding but had abrogated oriP-dependent DNA replication. We also observed that the AT-rich intervening DNA between EBNA1 binding sites in the FR can be occupied by the EBNA1 AT hook, N-terminal domain (NTD) aa 1 to 90 to form a Zn-dependent stable complex with EBNA1 DBD on a 2×FR DNA template. We propose a model showing EBNA1 DBD and NTD cobinding at the FR and suggest that this may contribute to the oligomerization of viral episomes important for maintenance during latent infection. IMPORTANCE EBV latent infection is causally linked to diverse cancers and autoimmune disorders. EBNA1 is the viral-encoded DNA binding protein required for episomal maintenance during latent infection and is consistently expressed in all EBV tumors. The interaction of EBNA1 with different genetic elements confers different viral functions, such as replication initiation at DS and chromosome tethering at FR. Here, we used cryo-EM to determine the structure of the EBNA1 DNA-binding domain (DBD) bound to two tandem sites at the DS and at the FR. We also show that the NTD of EBNA1 can interact with the AT-rich DNA sequence between tandem EBNA1 DBD binding sites in the FR. These results provide new information on the mechanism of EBNA1 DNA binding at DS and FR and suggest a higher-order oligomeric structure of EBNA1 bound to FR. Our findings have implications for targeting EBNA1 in EBV-associated disease.

WGS CHARACTERIZATION OF ENTEROCOCCUS FAECALIS H1041 ISOLATED FROM THE TRADITIONAL BULGARIAN GREEN CHEESE

In this study, Enterococcus faecalis H1041 which was isolated from the traditional Bulgarian green cheese was characterized in depth by a whole-genome sequencing performed on the Illumina HiSeq platform using 2 x 150 bases paired-end reads mode. The bioinformatic analysis performed only with free online tools revealed that the strain resembles other cheese-born E. faecalis strains by its antibiotics’ resistances and virulence genotype, thus its presence within the green cheese should not be considered as a health risk. Only two antibiotic resistance genetic determinants were found, but they both carry mutations, and probably are not functional. Furthermore, 18 virulence factor genetic determinants were found, but only 6 of them possessed 100% identities with the canonical sequences, and being more or less intrinsic for the Enterococcus genus. None of them were found on contigs that contain sequences with known plasmid origin of replication. Enterolysin A gene was also detected which most probably plays role in ripening process of the Bulgarian green cheese.

Ubiquitous Conjugative Mega-Plasmids of Acinetobacter Species and Their Role in Horizontal Transfer of Multi-Drug Resistance.

Conjugative mega-plasmids play a special role in adaptation since they carry a huge number of accessory genes, often allowing the host to develop in new niches. In addition, due to conjugation they are able to effectively spread themselves and participate in the transfer of small mobilizable plasmids. In this work, we present a detailed characterization of a recently discovered family of multiple-drug resistance mega-plasmids of Acinetobacter species, termed group III-4a. We describe the structure of the plasmid backbone region, identify the rep gene and the origin of plasmid replication, and show that plasmids from this group are able not only to move between different Acinetobacter species but also to efficiently mobilize small plasmids containing different mob genes. Furthermore, we show that the population of natural Acinetobacter strains contains a significant number of mega-plasmids and reveal a clear correlation between the living conditions of Acinetobacter strains and the structure of their mega-plasmids. In particular, comparison of the plasmids from environmental and clinical strains shows that the genes for resistance to heavy metals were eliminated in the latter, with the simultaneous accumulation of antibiotic resistance genes by incorporation of transposons and integrons carrying these genes. The results demonstrate that this group of mega-plasmids plays a key role in the dissemination of multi-drug resistance among Acinetobacter species.

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens.

Vibrio natriegens is known as the world's fastest growing organism with a doubling time of less than 10 min. This incredible growth speed empowers V.natriegens as a chassis for synthetic and molecular biology, potentially replacing E.coli in many applications. While first genetic parts have been built and tested for V.natriegens, a comprehensive toolkit containing well-characterized and standardized parts did not exist. To close this gap, we created the Marburg Collection-a highly flexible Golden Gate cloning toolbox optimized for the emerging chassis organism V.natriegens, containing 191 genetic parts. The Marburg Collection overcomes the paradigm of plasmid construction-integrating inserts into a backbone-by enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V.natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organism-specific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V.natriegens community.

Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA.

An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA–protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.

Mechanisms of Theta Plasmid Replication in Enterobacteria and Implications for Adaptation to Its Host.

Plasmids are autonomously replicating sequences that help cells adapt to diverse stresses. Theta plasmids are the most frequent plasmid class in enterobacteria. They co-opt two host replication mechanisms: replication at oriC, a DnaA-dependent pathway leading to replisome assembly (theta class A), and replication fork restart, a PriA-dependent pathway leading to primosome assembly through primer extension and D-loop formation (theta classes B, C, and D). To ensure autonomy from the host's replication and to facilitate copy number regulation, theta plasmids have unique mechanisms of replication initiation at the plasmid origin of replication (ori). Tight plasmid copy number regulation is essential because of the major and direct impact plasmid gene dosage has on gene expression. The timing of plasmid replication and segregation are also critical for optimizing plasmid gene expression. Therefore, we propose that plasmid replication needs to be understood in its biological context, where complex origins of replication (redundant origins, mosaic and cointegrated replicons), plasmid segregation, and toxin-antitoxin systems are often present. Highlighting their tight functional integration with ori function, we show that both partition and toxin-antitoxin systems tend to be encoded in close physical proximity to the ori in a large collection of Escherichia coli plasmids. We also propose that adaptation of plasmids to their host optimizes their contribution to the host's fitness while restricting access to broad genetic diversity, and we argue that this trade-off between adaptation to host and access to genetic diversity is likely a determinant factor shaping the distribution of replicons in populations of enterobacteria.

The large plasmidome of Lactococcus lactis subsp. lactis bv. diacetylactis S50 confers its biotechnological properties

Plasmids are autonomous episomally replicating genetic elements, which carry backbone genes important for the replication and maintenance within their host, and accessory genes that might confer an advantage to their host under specific selective pressure in its ecological niche. The genome of dairy isolate L. lactis subsp. lactis bv. diacetylactis S50 was sequenced using the PacBio SMRT Cell Seq-RSII platform and revealed to possess one of the largest plasmidomes among L. lactis strains studied so far, harboring six plasmids: pS6 (5553 bp), pS7a (7308 bp), pS7b (7266 bp), pS19 (19,027 bp), pS74 (74,256 bp) and pS127 (127,002 bp) in total representing 8.9% of genome size (240,412 bp). Based on predicted plasmid replication proteins and origins it appears that all six plasmids replicate via the theta-type mechanism. The two the largest plasmids (pS74 and pS127), carry a number of genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as bacteriocin production, protein degradation, magnesium and cobalt/nickel transporters, selenium binding, exopolysaccharides (EPS) production, bacteriophage and stress resistance. Beside genes for replication, the small plasmids (pS6, pS7a, pS7a, and pS19) also carry genes important for mobilization and host survival such as type I restriction-modification (R-M) system, metal transporters, enzymes and transcriptional regulators. All plasmids in S50 strain are mobilizable, containing an oriT sequences, while pS127 is self-conjugative and allows for mobilization of the other plasmids. Small plasmids are prone to structural and segregational instability, while pS127 appeared to be segregationally stable thanks to the possession of two partition systems. The main characteristic of plasmid pS74 is EPS production, while plasmid pS127 is characterized by proteinase and multiple bacteriocins, tra locus, phage abortive systems and metal transporters. In addition to LcnA and LcnB, plasmid pS127 encodes several bacteriocin-pheromone molecules and a new bacteriocin named LcnS50, with narrow spectrum of action limited to lactococci, that has been successfully cloned and heterologously expressed.

Prolonged siRNA expression in mammalian cells using an Epstein–Barr virus-based plasmid expression system

RNA interference (RNAi) is a powerful tool in gene function analysis and disease treatment, especially diseases that are ‘undruggable’ by classical small molecules. However, the RNAi applications are limited due to some defects, such as short duration and toxic side effects. New strategies are still needed to improve RNAi applications. Previous studies have illustrated that Epstein-Barr virus nuclear antigen 1 (EBNA-1) and the origin of plasmid replication (oriP) are critical factors for EBV latent gene expression, which can keep the replication of the EBV genome as an extrachromosomal element for a relatively long time. Here we report a plasmid expression system on the base of oriP and EBNA-1, which could produce protein as well as short interfering RNAs(siRNAs) for a long time in mammalian cells. siRNA expression mediated by this system causes efficient and specific down-regulation of gene expression. Except for analyzing gene function, this study also provided a new optional and practical way for protein and/or RNAi-based therapies that require enduring effect.

Molecular dissection of the replication system of plasmid pIGRK encoding two in-frame Rep proteins with antagonistic functions

BackgroundGene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called “trivial overlapping”, there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome’s novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family.ResultsA single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR’ protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR’ have opposing functions – RepR is crucial for initiation of pIGRK replication, while RepR’ is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression.ConclusionsRegulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

Structural Basis for Cooperative Binding of EBNA1 to the Epstein-Barr Virus Dyad Symmetry Minimal Origin of Replication.

Epstein-Barr virus is associated with several human malignancies, including nasopharyngeal carcinoma, gastric cancer, and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (oriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half-site. This structure shows that the DNA is smoothly bent, allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised oriP-containing plasmid replication in cells, and impaired recruitment of the MCM3 complex to the oriP Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the oriP Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.IMPORTANCE Epstein-Barr virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of Epstein-Barr virus nuclear antigen 1 (EBNA1) with the viral origin of plasmid replication (oriP). The dyad symmetry (DS) element in oriP is the essential minimal replicator of oriP Here we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.

Heavy metal resistance genes and plasmid-mediated quinolone resistance genes in Arthrobacter sp. isolated from Brazilian soils.

Arthrobacter sp. are Gram-positive bacilli commonly obtained from soil and in the hospital environment. These species have been reported to cause several types of infection. Heavy metals are a threat to the ecological system due to their high-levels of toxicity and the fluoroquinolones are antimicrobials widely used for the treatment of different bacterial infections. The aim of this study was to investigate the resistance to fluoroquinolone and heavy metals, the presence of plasmid-mediated resistance (PMQR) genes and heavy metals resistance (HMR) genes and the presence of plasmids in Arthrobacter sp. obtained from Brazilian soils. Bacterial isolation was performed using soil samples from different Brazilian regions. The bacterial identification was performed by 16S rRNA gene sequencing. The resistance profile for fluoroquinolones and heavy metals was determined by MIC. Several PMQR and HMR genes and plasmid families were investigated by PCR. Eight isolates were obtained from soil samples from different cultivations and regions of Brazil. All isolates were resistant to all fluoroquinolones, cadmium, cobalt and zinc and the majority to copper. Among the PMQR genes, the qepA (4) was the most prevalent, followed by qnrS (3), qnrB (3), oqxB (2) and oqxA (1). Among the HMR genes, the copA was detected in all isolates and the czcA in two isolates. The replication origin of the ColE-like plasmid was detected in all isolates; however, no plasmid was detected by extraction. The association of resistance to heavy metals and antimicrobials is a threat to the environmental balance and to human health. There are no studies reporting the association of PMQR and HMR genes in bacteria belonging to the genus Arthrobacter. To the best of our knowledge, this is the first report of qnrB, qepA, oqxA and oqxB in Arthrobacter species.

Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in Candida albicans.

The ability to generate autonomously replicating plasmids has been elusive in Candida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively). Candida albicansURA3 and a heterologous marker, ARG4, yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologous LEU2 marker was highly dependent upon the addition of a bona fide origin sequence. Several bona fide chromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin, panARS, from Kluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, C. albicans maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.IMPORTANCE Circular plasmids are important tools for molecular manipulation in model fungi such as baker's yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a C. albicans origin and an origin sequence from a distantly related yeast can both function as origins in C. albicans Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and baker's yeast.

Epstein-Barr Virus Genome Replication as a Molecular Target for Cancer Therapy

Epstein-Barr virus (EBV), a human oncogenic virus, is a B cell-tropic herpesvirus and has the ability to immortalize normal B cells during latent infection. The Epstein-Barr nuclear antigen 1 (EBNA1) protein of EBV is expressed in the most EBV latently infected cells and binds to a specific viral genome region termed "oriP" (origin of plasmid replication) to maintain the stability of the approximately 170 kb double-stranded circular virus genomic DNA (episome) in cells. EBV elimination is thought to inhibit progression of EBV-associated malignancies, and the EBNA1-dependent mechanisms for EBV episome replication and maintenance are considered to be novel molecular targets for anti-EBV therapy. We have explored small-molecule compounds that can inhibit the binding between EBNA1 protein and oriP and found one pyrrole imidazole polyamide named DSE3 which can also inhibit EBV-mediated immortalization of normal B cells. These data suggested that an EBNA1-targeting strategy could be useful to combat EBV-associated malignancies.

DoriC 10.0: an updated database of replication origins in prokaryotic genomes including chromosomes and plasmids.

DoriC, a database of replication origins, was initially created to present the bacterial oriCs predicted by Ori-Finder or determined by experiments in 2007. DoriC 5.0, an updated database of oriC regions in both bacterial and archaeal genomes, was published in the 2013 Nucleic Acids Research database issue. Now, the latest release DoriC 10, a large-scale update of replication origins in prokaryotic genomes including chromosomes and plasmids, has been presented with a completely redesigned user interface, which is freely available at http://tubic.org/doric/ and http://tubic.tju.edu.cn/doric/. In the current release, the database of DoriC has made significant improvements compared with version 5.0 as follows: (i) inclusion of oriCs on more bacterial chromosomes increased from 1633 to 7580; (ii) inclusion of oriCs on more archaeal chromosomes increased from 86 to 226; (iii) inclusion of 1209 plasmid replication origins retrieved from NCBI annotations or predicted by in silico analysis; (iv) inclusion of more replication origin elements on bacterial chromosomes including DnaA-trio motifs. Now, DoriC becomes the most complete and scalable database of replication origins in prokaryotic genomes, and facilitates the studies in large-scale oriC data mining, strand-biased analyses and replication origin predictions.

Novel Pyrrole–Imidazole Polyamide Hoechst Conjugate Suppresses Epstein–Barr Virus Replication and Virus-Positive Tumor Growth

Epstein-Barr virus (EBV) establishes latent infection and is associated with several types of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV-positive tumor cells. EBNA1 binds to the origin of virus plasmid replication (OriP) on the EBV episome to initiate virus DNA replication and regulates virus gene expression as a transcriptional activator. In this study, we designed and synthesized a pyrrole-imidazole polyamide-Hoechst 33258 conjugate named EIP-2 (2), which specifically binds to the OriP region with high affinity, to interrupt EBNA1-OriP binding in vitro and in vivo. By eradicating the EBV episome in EBV-positive cells, compound 2 selectively inhibited EBV-positive cell proliferation. Moreover, the injection of 2 significantly suppressed tumor growth in the mice xenograft tumor model. These findings demonstrate that compound 2 is a potential therapeutic candidate for the treatment of EBV-associated tumors.