IN VIVO INTERMEDIATES AND THE ROLLING CIRCLE MECHANISM IN VIROID REPLICATION

ABSTRACT Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2′,5′-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2′,5′-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2′,5′-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2′,5′-phosphodiester bonds are still present in nature and that they are of biological importance.

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Viroids: unusual small pathogenic RNAs.

Acta Biochimica Polonica ◽

10.18388/abp.2004_3546 ◽

2004 ◽

Vol 51 (3) ◽

pp. 587-607 ◽

Cited By ~ 38

Author(s):

Anna Góra-Sochacka

Keyword(s):

Secondary Structure ◽

Hammerhead Ribozyme ◽

Direct Interaction ◽

Plant Diseases ◽

Plant Host ◽

Rolling Circle ◽

Rna Molecules ◽

Viroid Replication ◽

Plant Genes ◽

Rolling Circle Mechanism

Viroids are small (about 300 nucleotides), single-stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Viroids of the Avsunviroidae family adopt a branched or quasi rod-like secondary structure in their native state. Members of the Pospiviroidae family adopt a rod-like secondary structure. In such native structures five structural/functional domains have been identified: central (C), pathogenicity, variable and two terminal domains. The central conserved region (CCR) within the C domain characterizes viroids of the Pospiviroidae. Specific secondary structures of this region play an important role in viroid replication and processing. Viroids of the Avsunviroidae family lack a CCR but possess self-cleaving properties by forming hammerhead ribozyme structures; they accumulate and replicate in chloroplasts, whereas members of the Pospiviroidae family have a nuclear localization. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation.

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Lagging-Strand Replication from the ssoA Origin of Plasmid pMV158 in Streptococcus pneumoniae: In Vivo and In Vitro Influences of Mutations in Two ConservedssoA Regions

Journal of Bacteriology ◽

10.1128/jb.180.1.83-89.1998 ◽

1998 ◽

Vol 180 (1) ◽

pp. 83-89 ◽

Cited By ~ 22

Author(s):

M. Gabriela Kramer ◽

Saleem A. Khan ◽

Manuel Espinosa

Keyword(s):

Streptococcus Pneumoniae ◽

Sequence Specificity ◽

Single Stranded Dna ◽

Rolling Circle ◽

Cell Extracts ◽

Rolling Circle Mechanism ◽

Derivative Plasmid ◽

Lagging Strand

ABSTRACT The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RSB] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication inStreptococcus pneumoniae. Cells containing plasmids with mutations in the RSB accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RSB region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.

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Monomeric Linear RNA of Citrus Exocortis Viroid Resulting from Processing In Vivo Has 5′-Phosphomonoester and 3′-Hydroxyl Termini: Implications for the RNase and RNA Ligase Involved in Replication

Journal of Virology ◽

10.1128/jvi.01229-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 10321-10325 ◽

Cited By ~ 25

Author(s):

María-Eugenia Gas ◽

Diego Molina-Serrano ◽

Carmen Hernández ◽

Ricardo Flores ◽

José-Antonio Daròs

Keyword(s):

Asymmetric Rolling ◽

Rolling Circle ◽

Rna Ligase ◽

Citrus Exocortis Viroid ◽

The Family ◽

Rolling Circle Mechanism ◽

Rapid Amplification ◽

Hydroxyl Termini

ABSTRACT Members of the family Pospiviroidae, like Citrus exocortis viroid (CEVd), replicate through an RNA-based asymmetric rolling-circle mechanism in which oligomeric plus-strand [(+)] RNA intermediates are cleaved to monomeric linear (ml) RNA and then circularized. Here we show, by rapid amplification of 5′ and 3′ cDNA ends and in vitro ligation assays, that ml CEVd (+) RNA resulting from cleavage of a dimeric transcript transgenically expressed in Arabidopsis thaliana contains 5′-phosphomonoester and 3′-hydroxyl termini. The nature of these termini and the double-stranded structure previously proposed as the substrate for cleavage in vivo suggest that a type III RNase catalyzes cleavage and an RNA ligase distinct from tRNA ligase promotes circularization.

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Autonomous Replication of the Conjugative Transposon Tn916

Journal of Bacteriology ◽

10.1128/jb.00639-16 ◽

2016 ◽

Vol 198 (24) ◽

pp. 3355-3366 ◽

Cited By ~ 21

Author(s):

Laurel D. Wright ◽

Alan D. Grossman

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Origin Of Replication ◽

Rolling Circle Replication ◽

Conjugative Transposon ◽

Rolling Circle ◽

Autonomous Replication ◽

Integrative And Conjugative Elements ◽

Rolling Circle Mechanism ◽

Origin Of Transfer

ABSTRACTIntegrative and conjugative elements (ICEs), also known as conjugative transposons, are self-transferable elements that are widely distributed among bacterial phyla and are important drivers of horizontal gene transfer. Many ICEs carry genes that confer antibiotic resistances to their host cells and are involved in the dissemination of these resistance genes. ICEs reside in host chromosomes but under certain conditions can excise to form a plasmid that is typically the substrate for transfer. A few ICEs are known to undergo autonomous replication following activation. However, it is not clear if autonomous replication is a general property of many ICEs. We found that Tn916, the first conjugative transposon identified, replicates autonomously via a rolling-circle mechanism. Replication of Tn916was dependent on the relaxase encoded byorf20of Tn916. The origin of transfer of Tn916,oriT(916), also functioned as an origin of replication. Using immunoprecipitation and mass spectrometry, we found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916likely interact in a complex and that the Tn916relaxase contains a previously unidentified conserved helix-turn-helix domain in its N-terminal region that is required for relaxase function and replication. Lastly, we identified a functional single-strand origin of replication (sso) in Tn916that we predict primes second-strand synthesis during rolling-circle replication. Together these results add to the emerging data that show that several ICEs replicate via a conserved, rolling-circle mechanism.IMPORTANCEIntegrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.

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rolling circle mechanism

Catalysis from A to Z ◽

10.1002/9783527809080.cataz14510 ◽

2020 ◽

Keyword(s):

Rolling Circle ◽

Rolling Circle Mechanism

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

Cells ◽

10.3390/cells9020467 ◽

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.

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Sequence Analysis of Plasmid pCC5.2 from CyanobacteriumSynechocystisPCC 6803 That Replicates by a Rolling Circle Mechanism

Plasmid ◽

10.1006/plas.1997.1281 ◽

1997 ◽

Vol 37 (2) ◽

pp. 95-104 ◽

Cited By ~ 14

Author(s):

Weidong Xu ◽

Bruce A. McFadden

Keyword(s):

Sequence Analysis ◽

Rolling Circle ◽

Rolling Circle Mechanism

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pBMSa1, a plasmid from a dairy cow isolate of Staphylococcus aureus, encodes a lincomycin resistance determinant and replicates by the rolling-circle mechanism

Plasmid ◽

10.1016/j.plasmid.2004.03.001 ◽

2004 ◽

Vol 52 (1) ◽

pp. 48-56 ◽

Cited By ~ 21

Author(s):

Pedro D Loeza-Lara ◽

Morelia Soto-Huipe ◽

Victor M Baizabal-Aguirre ◽

Alejandra Ochoa-Zarzosa ◽

Juan J Valdez-Alarcón ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Dairy Cow ◽

Resistance Determinant ◽

Rolling Circle ◽

Rolling Circle Mechanism ◽

Lincomycin Resistance

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Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo

Journal of Virology ◽

10.1128/jvi.74.20.9451-9463.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9451-9463 ◽

Cited By ~ 144

Author(s):

Hiroyuki Nakai ◽

Theresa A. Storm ◽

Mark A. Kay

Keyword(s):

Rolling Circle Replication ◽

Chromosomal Dna ◽

Adeno Associated Virus ◽

Rolling Circle ◽

Steady State Levels ◽

Slow Rise ◽

Mouse Hepatocytes ◽

Enzyme Recognition

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

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