scholarly journals Rolling-Circle Replication of an Animal Circovirus Genome in a Theta-Replicating Bacterial Plasmid in Escherichia coli

2006 ◽  
Vol 80 (17) ◽  
pp. 8686-8694 ◽  
Author(s):  
Andrew K. Cheung
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Unit Length ◽  
Release Mechanism ◽  
Rolling Circle Replication ◽  
Replication Process ◽  
Rolling Circle ◽  
Rep Protein ◽  
Bacterial Plasmid

ABSTRACT A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1Rep/PCV2Cap genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1Cap/PCV2Rep genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE1 Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep′ protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep′ protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.

Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses

2021 ◽  
Vol 102 (11) ◽  
Author(s):  
Rafaela S. Fontenele ◽  
Matias Köhler ◽  
Lucas C. Majure ◽  
Jesús A. Avalos-Calleros ◽  
Gerardo R. Argüello-Astorga ◽  
...  
Keyword(s):  
South America ◽  
Rolling Circle Replication ◽  
Dna Virus ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Circular Dna ◽  
Rolling Circle ◽  
Rep Protein ◽  
Molecular Features ◽  
Viral Components

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

scholarly journals Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 467
Author(s):  
Min Hao ◽  
Zhaoguan Wang ◽  
Hongyan Qiao ◽  
Peng Yin ◽  
Jianjun Qiao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
Genome Engineering ◽  
Single Gene ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Cas9 Nuclease ◽  
Dynamic Genome ◽  
Circular Ssdna

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

scholarly journals Genetic and Biochemical Characterization of the Streptococcus pneumoniae PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

2006 ◽  
Vol 188 (21) ◽  
pp. 7416-7425 ◽  
Author(s):  
J. A. Ruiz-Masó ◽  
S. P. Anand ◽  
M. Espinosa ◽  
S. A. Khan ◽  
G. del Solar
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biochemical Characterization ◽  
Essential Gene ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Rolling Circle Replication ◽  
Lower Efficiency ◽  
Rolling Circle ◽  
Rep Protein

ABSTRACT PcrA is a chromosomally encoded DNA helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. Efficient interaction between PcrA and the plasmid-encoded replication initiator (Rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a Rep protein to interact with heterologous PcrA helicases has been invoked as a determinant of plasmid promiscuity. We characterized transcription of the Streptococcus pneumoniae pcrA gene in its genetic context and studied the biochemical properties of its product, the PcrA Spn helicase. Transcription of the pneumococcal pcrA gene was directed by promoter Pa, consisting of an extended −10 box. Promoter Pa also accounted for expression of a second essential gene, radC, which was transcribed with much lower efficiency than pcrA, probably due to the presence of a terminator/attenuator sequence located between the two genes. PcrA Spn displayed single-stranded DNA-dependent ATPase activity. PcrA Spn showed 5′→3′ and 3′→5′ helicase activities and bound efficiently to partially duplex DNA containing a hairpin structure adjacent to a 6-nucleotide 5′ or 3′ single-stranded tail and one unpaired (flap) nucleotide in the complementary strand. PcrA Spn interacted specifically with RepC, the initiator of staphylococcal plasmid pT181. Although the pneumococcal helicase was able to initiate unwinding of the RepC-nicked pT181 DNA, it was much less processive in this activity than the cognate staphylococcal PcrA protein. Accordingly, PcrA Spn was inefficient in in vitro replication of pT181, and perhaps as a consequence, this plasmid could not be established in S. pneumoniae.

scholarly journals A transcription and translation-coupled DNA replication system using rolling-circle replication

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Yoshihiro Sakatani ◽  
Norikazu Ichihashi ◽  
Yasuaki Kazuta ◽  
Tetsuya Yomo
Keyword(s):  
Dna Replication ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Replication System

scholarly journals A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues

Viruses ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 37
Author(s):  
Elvira Tarasova ◽  
Reza Khayat
Keyword(s):  
Structural Information ◽  
Experimental Studies ◽  
Structural Data ◽  
Rolling Circle Replication ◽  
Dna Viruses ◽  
Rolling Circle ◽  
Structural Mechanism ◽  
Functional Sites ◽  
Bacterial Plasmid ◽  
Shed Light

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

scholarly journals Self-assembly Controls Self-cleavage of HHR from ASBVd(-): a Combined SANS and Modeling Study.

2016 ◽  
Author(s):  
Fabrice Leclerc ◽  
Giuseppe Zaccai ◽  
Jacques Vergne ◽  
Martina Rihova ◽  
Anne Martel ◽  
...  
Keyword(s):  
Self Assembly ◽  
Unit Length ◽  
3D Models ◽  
Active Conformation ◽  
Rolling Circle Replication ◽  
Hammerhead Ribozymes ◽  
Temperature Dependent ◽  
Rolling Circle ◽  
Rate Determining Step ◽  
2D And 3D

In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.

scholarly journals Analysis of transcription of Porcine circovirus type 1

2002 ◽  
Vol 83 (11) ◽  
pp. 2743-2751 ◽  
Author(s):  
Annette Mankertz ◽  
Bernd Hillenbrand
Keyword(s):  
Infectious Clone ◽  
Porcine Circovirus Type ◽  
Open Reading Frames ◽  
Porcine Circovirus ◽  
Origin Of Replication ◽  
Rolling Circle Replication ◽  
Structural Protein ◽  
Rolling Circle ◽  
Rep Protein

Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication initiator proteins, Rep and Rep′, and the structural protein, Cap. The promoters of these two genes (P cap and P rep ) have been mapped. P cap is located within the rep open reading frame (nt 1328–1252). P rep has been mapped to the intergenic region immediately upstream of the rep gene (nt 640–796) and overlaps the origin of replication of PCV1. Although binding of both rep gene products to a fragment containing P rep and the overlapping origin of replication has been reported, only the full-length Rep protein repressed P rep , while the spliced isoform Rep′ did not. P rep repression is mediated by binding of the Rep protein to the two inner hexamers, H1 and H2, located in the origin of PCV1, whereas binding of Rep to hexamers H3 and H4 was not necessary. Use of Rep mutants indicated that the conserved rolling-circle replication domain II as well as the P loop are essential for repression of P rep . In contrast to P rep , transcription of P cap was not influenced by viral proteins. Additionally, the ratio of the rep and rep′ transcripts was analysed. Twelve hours after transfection of PK15 cells with an infectious clone of PCV1, similar amounts of both transcripts were detected, but later the amount of the two transcripts varied, indicating a balanced expression of the two rep transcripts.

scholarly journals Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

2004 ◽  
Vol 70 (12) ◽  
pp. 6957-6962 ◽  
Author(s):  
Gilad Bachrach ◽  
Susan Kinder Haake ◽  
Alon Glick ◽  
Ronen Hazan ◽  
Ronit Naor ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Plasmid Stability ◽  
Protein A ◽  
Fusobacterium Nucleatum ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Rep Protein ◽  
Shuttle Plasmid ◽  
Plasmid Addiction System

ABSTRACT Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.

scholarly journals Detection of Template Strand Switching during Initiation and Termination of DNA Replication of Porcine Circovirus

2004 ◽  
Vol 78 (8) ◽  
pp. 4268-4277 ◽  
Author(s):  
Andrew K. Cheung
Keyword(s):  
Dna Replication ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Inverted Repeats ◽  
Minus Strand ◽  
Gene Correction ◽  
Melting Pot ◽  
Rolling Circle ◽  
Rep Protein ◽  
Template Strand

ABSTRACT 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.

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