scholarly journals Lagging-Strand DNA Replication Origins Are Required for Conjugal Transfer of the Promiscuous Plasmid pMV158

2008 ◽  
Vol 191 (3) ◽  
pp. 720-727 ◽  
Author(s):  
Fabián Lorenzo-Díaz ◽  
Manuel Espinosa
Keyword(s):  
Streptococcus Pneumoniae ◽  
Plasmid Dna ◽  
Essential Role ◽  
Critical Factor ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
New Hosts ◽  
Rolling Circle Mechanism ◽  
Dna Replication Origins ◽  
Lagging Strand

ABSTRACT The promiscuous streptococcal plasmid pMV158 is mobilizable by auxiliary plasmids and replicates by the rolling-circle mechanism in a variety of bacterial hosts. The plasmid has two lagging-strand origins, ssoA and ssoU, involved in the conversion of single-stranded DNA intermediates into double-stranded plasmid DNA during vegetative replication. Transfer of the plasmid also would involve conversion of single-stranded DNA molecules into double-stranded plasmid forms in the recipient cells by conjugative replication. To test whether lagging-strand origins played a role in horizontal transfer, pMV158 derivatives defective in one or in both sso's were constructed and tested for their ability to colonize new hosts by means of intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae, only plasmids that had an intact ssoU could be efficiently mobilized from S. pneumoniae to Enterococcus faecalis. Thus, it appears that ssoU is a critical factor for pMV158 promiscuity and that the presence of a functional sso plays an essential role in plasmid transfer.

scholarly journals 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

1998 ◽  
Vol 180 (1) ◽  
pp. 83-89 ◽  
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.

scholarly journals An iterated sequence in the genome of Banana bunchy top virus is essential for efficient replication

2006 ◽  
Vol 87 (11) ◽  
pp. 3409-3412 ◽  
Author(s):  
Virginia A. Herrera-Valencia ◽  
Benjamin Dugdale ◽  
Robert M. Harding ◽  
James L. Dale
Keyword(s):  
Intergenic Region ◽  
Viral Genome ◽  
Specific Interaction ◽  
Replication Initiation ◽  
Banana Bunchy Top Virus ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
Replication Initiation Protein ◽  
Efficient Replication ◽  
Rolling Circle Mechanism

Banana bunchy top virus (BBTV) has a multi-component genome of circular, single-stranded DNA. BBTV replicates via a rolling-circle mechanism, probably involving sequence-specific interaction of the replication initiation protein (Rep) with iterated sequences (iterons) within the viral genome. Three putative iterons (designated F1, F2 and R), with the sequence GGGAC, have been identified in the intergenic region of each BBTV component. To investigate their role in replication, each of the iterons was mutated, singularly and in tandem, in a BBTV DNA-N 1.1mer and the ability of these molecules to be replicated by the BBTV ‘master’ Rep was evaluated in banana cells using transient biolistic assays. All iteron mutants were replicated less efficiently than the native DNA-N. Mutation of the F1 and R iterons caused a 42 and 62 % reduction in DNA-N replication, respectively, whereas mutation of the F2 and combined F1F2 iteron virtually abolished DNA-N replication.

scholarly journals Characterization of Plasmid pRT1 from Pyrococcus sp. Strain JT1

2002 ◽  
Vol 184 (9) ◽  
pp. 2561-2566 ◽  
Author(s):  
Donald E. Ward ◽  
Ingrid M. Revet ◽  
Renu Nandakumar ◽  
Jon H. Tuttle ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
Dna Replication ◽  
Open Reading Frames ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
Hyperthermophilic Archaeon ◽  
Rolling Circle Mechanism ◽  
Replication Intermediates ◽  
Reading Frames

ABSTRACT We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon Pyrococcus sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.

scholarly journals Sinorhizobium meliloti Plasmid pRm1132f Replicates by a Rolling-Circle Mechanism

2001 ◽  
Vol 183 (8) ◽  
pp. 2704-2708 ◽  
Author(s):  
L. R. Barran ◽  
N. Ritchot ◽  
E. S. P. Bromfield
Keyword(s):  
Nucleotide Sequence ◽  
Plasmid Dna ◽  
Sinorhizobium Meliloti ◽  
Open Reading Frames ◽  
Rolling Circle ◽  
Group Iii ◽  
Coding Regions ◽  
Origins Of Replication ◽  
Rolling Circle Mechanism ◽  
Reading Frames

ABSTRACT pRm1132f isolated from Sinorhizobium meliloti is a group III rolling-circle-replicating (RCR) plasmid. At least seven of eight open reading frames in the nucleotide sequence represented coding regions. The minimal replicon contained a rep gene and single- and double-stranded origins of replication. Detection of single-stranded plasmid DNA confirmed that pRm1132f replicated via an RCR mechanism.

scholarly journals ICTV Virus Taxonomy Profile: Plectroviridae

2021 ◽  
Vol 102 (5) ◽  
Author(s):  
Petar Knezevic ◽  
Evelien M. Adriaenssens ◽  
Keyword(s):  
Cell Wall ◽  
International Committee ◽  
Virus Taxonomy ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
Bacterial Surface ◽  
Link Type ◽  
Rigid Rods ◽  
The Family ◽  
Rolling Circle Mechanism

Members of the family Plectroviridae produce particles that are non-enveloped rigid rods (70–280×10–16 nm). The supercoiled, circular, single-stranded DNA genome of about 4.5–8.3 kb, encodes 4–13 proteins. Viruses of this family infect cell wall-less bacteria, adsorbing to the bacterial surface, replicating their DNA by a rolling-circle mechanism or transposition, and releasing progeny from cells by extrusion, without killing the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Plectroviridae which is available at ictv.global/report/plectroviridae.

scholarly journals Natural 2′,5′-Phosphodiester Bonds Found at the Ligation Sites of Peach Latent Mosaic Viroid

2001 ◽  
Vol 75 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Fabien Côté ◽  
Dominique Lévesque ◽  
Jean-Pierre Perreault
Keyword(s):  
Circular Rna ◽  
Rolling Circle ◽  
Phosphodiester Bonds ◽  
Biological Importance ◽  
Rolling Circle Mechanism ◽  
Ligation Site

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.

scholarly journals Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27

1999 ◽  
Vol 19 (6) ◽  
pp. 4143-4152 ◽  
Author(s):  
Julie Parenteau ◽  
Raymund J. Wellinger
Keyword(s):  
Growth Arrest ◽  
Dna Lesions ◽  
Restrictive Temperature ◽  
Telomeric Dna ◽  
Single Stranded Dna ◽  
Template Strand ◽  
Okazaki Fragments ◽  
A Cell ◽  
Lagging Strand

ABSTRACT The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the processing of Okazaki fragments during DNA lagging-strand synthesis. One of the predicted DNA lesions occurring in rad27 strains is the presence of single-stranded DNA of the template strand for lagging-strand synthesis. We examined this prediction by analyzing the terminal DNA structures generated during telomere replication in rad27strains. The lengths of the telomeric repeat tracts were found to be destabilized in rad27 strains, indicating that naturally occurring direct repeats are subject to tract expansions and contractions in such strains. Furthermore, abnormally high levels of single-stranded DNA of the templating strand for lagging-strand synthesis were observed in rad27 cells. Overexpression of Dna2p in wild-type cells also yielded single-stranded DNA regions on telomeric DNA and caused a cell growth arrest phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature. Furthermore, overexpression of the yeast exonuclease Exo1p alleviated the growth arrest induced by both conditions, overexpression of Dna2p and incubation of rad27cells at 37°C. However, the telomere heterogeneity and the appearance of single-stranded DNA are not prevented by the overexpression of Exo1p in these strains, suggesting that this nuclease is not simply redundant with Rad27p. Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging-strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.

scholarly journals An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

2020 ◽  
Author(s):  
Catherine D. Aimone ◽  
J. Steen Hoyer ◽  
Anna E. Dye ◽  
David O. Deppong ◽  
Siobain Duffy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rolling Circle Amplification ◽  
Next Generation ◽  
Viral Dna ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Short Read ◽  
Rolling Circle ◽  
Optimized Protocol ◽  
Generation Sequencing

AbstractWe present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.HighlightsProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primersComparison of the sequencing of total DNA versus size-selected DNAComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes

scholarly journals Viroids: unusual small pathogenic RNAs.

2004 ◽  
Vol 51 (3) ◽  
pp. 587-607 ◽  
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.

scholarly journals Autonomous Replication of the Conjugative Transposon Tn916

2016 ◽  
Vol 198 (24) ◽  
pp. 3355-3366 ◽  
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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