scholarly journals Genome replication dynamics of a bacteriophage and its satellite reveal strategies for parasitism and viral restriction

2019 ◽  
Author(s):  
Zachary K Barth ◽  
Tania V Silvas ◽  
Angus Angermeyer ◽  
Kimberley D Seed
Keyword(s):  
Dna Replication ◽  
Vibrio Cholerae ◽  
Host Cells ◽  
Initiation Factor ◽  
Cell Populations ◽  
Lytic Phage ◽  
Genome Replication ◽  
Origin Of Replication ◽  
New Host ◽  
Viral Restriction

Abstract Phage-inducible chromosomal island-like elements (PLEs) are bacteriophage satellites found in Vibrio cholerae. PLEs parasitize the lytic phage ICP1, excising from the bacterial chromosome, replicating, and mobilizing to new host cells following cell lysis. PLEs protect their host cell populations by completely restricting the production of ICP1 progeny. Previously, it was found that ICP1 replication was reduced during PLE(+) infection. Despite robust replication of the PLE genome, relatively few transducing units are produced. We investigated if PLE DNA replication itself is antagonistic to ICP1 replication. Here we identify key constituents of PLE replication and assess their role in interference of ICP1. PLE encodes a RepA_N initiation factor that is sufficient to drive replication from the PLE origin of replication during ICP1 infection. In contrast to previously characterized bacteriophage satellites, expression of the PLE initiation factor was not sufficient for PLE replication in the absence of phage. Replication of PLE was necessary for interference of ICP1 DNA replication, but replication of a minimalized PLE replicon was not sufficient for ICP1 DNA replication interference. Despite restoration of ICP1 DNA replication, non-replicating PLE remained broadly inhibitory against ICP1. These results suggest that PLE DNA replication is one of multiple mechanisms contributing to ICP1 restriction.

scholarly journals Genome replication dynamics of a bacteriophage and its satellite reveal strategies for parasitism and viral restriction

2019 ◽  
Author(s):  
Zachary K Barth ◽  
Tania V Silvas ◽  
Angus Angermeyer ◽  
Kimberley D Seed
Keyword(s):  
Dna Replication ◽  
Vibrio Cholerae ◽  
Host Cells ◽  
Initiation Factor ◽  
Cell Populations ◽  
Lytic Phage ◽  
Genome Replication ◽  
Origin Of Replication ◽  
New Host ◽  
Viral Restriction

ABSTRACTPhage-inducible chromosomal island-like elements (PLEs) are bacteriophage satellites found inVibrio cholerae. PLEs parasitize the lytic phage ICP1, excising from the bacterial chromosome, replicating, and mobilizing to new host cells following cell lysis. PLEs protect their host cell populations by completely restricting the production of ICP1 progeny. Previously, it was found that ICP1 replication was reduced during PLE(+) infection. Despite robustly replicating its genome, PLE produces relatively few transducing units, leading us to investigate if PLE DNA replication itself is antagonistic to ICP1 replication. Here we identify key constituents of PLE replication and assess their role in interference of ICP1. PLE encodes a RepA_N initiation factor that is sufficient to drive replication from the PLE origin of replication during ICP1 infection. In contrast to previously characterized bacteriophage satellites, expression of the PLE initiation factor was not sufficient for PLE replication in the absence of phage. Replication of PLE was necessary for interference of ICP1 DNA replication, but replication of a minimalized PLE replicon was not sufficient for ICP1 DNA replication interference. Despite restoration of ICP1 DNA replication, non-replicating PLE remained broadly inhibitory against ICP1. These results suggest that PLE DNA replication is one of multiple mechanisms contributing to ICP1 restriction.

scholarly journals The Stringent Response Inhibits DNA Replication Initiation inE. coliby Modulating Supercoiling oforiC

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
James A. Kraemer ◽  
Allen G. Sanderlin ◽  
Michael T. Laub
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Stringent Response ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Cell Physiology ◽  
Origin Of Replication ◽  
Content Type ◽  
Dna Replication Initiation ◽  
Negative Supercoils

ABSTRACTThe stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation inEscherichia coliremains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factordnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication,oriC. Active transcription normally introduces negative supercoils intooriCto help promote replication initiation, so the accumulation of ppGpp reduces initiation potential atoriCby reducing transcription. We find that maintaining transcription nearoriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein geneseqAalso relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils intooriC.IMPORTANCETo survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation inEscherichia colifor decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

scholarly journals Developmental differences in genome replication program and origin activation

2020 ◽  
Vol 48 (22) ◽  
pp. 12751-12777
Author(s):  
Cathia Rausch ◽  
Patrick Weber ◽  
Paulina Prorok ◽  
David Hörl ◽  
Andreas Maiser ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Embryonic Stem ◽  
S Phase ◽  
Developmental Differences ◽  
Centromeric Heterochromatin ◽  
Genome Replication ◽  
Origin Activation ◽  
Spatio Temporal ◽  
Mes Cells

Abstract To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.

scholarly journals Cell Death Signaling Pathway Induced by Cholix Toxin, a Cytotoxin and eEF2 ADP-Ribosyltransferase Produced by Vibrio cholerae

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 12
Author(s):  
Kohei Ogura ◽  
Kinnosuke Yahiro ◽  
Joel Moss
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Vibrio Cholerae ◽  
Elongation Factor ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Pseudomonas Exotoxin A ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Adp Ribosyltransferase

Pathogenic microorganisms produce various virulence factors, e.g., enzymes, cytotoxins, effectors, which trigger development of pathologies in infectious diseases. Cholera toxin (CT) produced by O1 and O139 serotypes of Vibrio cholerae (V. cholerae) is a major cytotoxin causing severe diarrhea. Cholix cytotoxin (Cholix) was identified as a novel eukaryotic elongation factor 2 (eEF2) adenosine-diphosphate (ADP)-ribosyltransferase produced mainly in non-O1/non-O139 V. cholerae. The function and role of Cholix in infectious disease caused by V. cholerae remain unknown. The crystal structure of Cholix is similar to Pseudomonas exotoxin A (PEA) which is composed of an N-terminal receptor-recognition domain and a C-terminal ADP-ribosyltransferase domain. The endocytosed Cholix catalyzes ADP-ribosylation of eEF2 in host cells and inhibits protein synthesis, resulting in cell death. In a mouse model, Cholix caused lethality with severe liver damage. In this review, we describe the mechanism underlying Cholix-induced cytotoxicity. Cholix-induced apoptosis was regulated by mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways, which dramatically enhanced tumor necrosis factor-α (TNF-α) production in human liver, as well as the amount of epithelial-like HepG2 cancer cells. In contrast, Cholix induced apoptosis in hepatocytes through a mitochondrial-dependent pathway, which was not stimulated by TNF-α. These findings suggest that sensitivity to Cholix depends on the target cell. A substantial amount of information on PEA is provided in order to compare/contrast this well-characterized mono-ADP-ribosyltransferase (mART) with Cholix.

scholarly journals The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

2005 ◽  
Vol 168 (7) ◽  
pp. 999-1012 ◽  
Author(s):  
Jeff Bachant ◽  
Shannon R. Jessen ◽  
Sarah E. Kavanaugh ◽  
Candida S. Fielding
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
S Phase ◽  
Origin Of Replication ◽  
Independent Manner ◽  
Checkpoint Signaling ◽  
Hydroxyurea Treatment ◽  
Chromatid Cohesion ◽  
S Phase Checkpoint

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

scholarly journals High Frequency of a Novel Filamentous Phage, VCYϕ, within an Environmental Vibrio cholerae Population

2011 ◽  
Vol 78 (1) ◽  
pp. 28-33 ◽  
Author(s):  
Hong Xue ◽  
Yan Xu ◽  
Yan Boucher ◽  
Martin F. Polz
Keyword(s):  
Vibrio Cholerae ◽  
Sequence Similarity ◽  
Host Population ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Filamentous Phage ◽  
Nucleotide Polymorphisms ◽  
Environmental Biology ◽  
Content Type ◽  
Filamentous Phages

ABSTRACTEnvironmentalVibrio choleraestrains isolated from a coastal brackish pond (Oyster Pond, Woods Hole, MA) carried a novel filamentous phage, VCYϕ, which can exist as a host genome integrative form (IF) and a plasmid-like replicative form (RF). Outside the cell, the phage displays a morphology typical ofInovirus, with filamentous particles ∼1.8 μm in length and 7 nm in width. Four independent RF isolates had identical genomes, except for 8 single nucleotide polymorphisms clustered in two regions. The overall genome size is 7,103 bp with 11 putative open reading frames organized into three functional modules (replication, structure and assembly, and regulation). VCYϕ shares sequence similarity with other filamentous phages (including cholera disease-associated CTX) in a highly mosaic manner, indicating evolution by horizontal gene transfer and recombination. VCYϕ integrates in the vicinity of the putative translation initiation factor Sui1 in chromosome II ofV. cholerae. A screen of 531 closely related host isolates showed that ∼40% harbored phages, with 27% and 13% carrying the IF and RF, respectively. The relative frequencies of the RF and IF differed among strains isolated from the pond or lagoon of Oyster Pond, suggesting that the host habitat influences intracellular phage biology. The overall high prevalence within the host population shows that filamentous phages can be an important component of the environmental biology ofV. cholerae.

scholarly journals Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms

2020 ◽  
Author(s):  
Y. Hu ◽  
A. Tareen ◽  
Y-J. Sheu ◽  
W. T. Ireland ◽  
C. Speck ◽  
...  
Keyword(s):  
Dna Replication ◽  
Gene Silencing ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Separate Analysis ◽  
Genome Replication ◽  
Sequence Specificity ◽  
Sequence Motifs ◽  
Origin Firing ◽  
Α Helix

AbstractDNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

scholarly journals Identification of unrecognized host factors promoting HIV-1 latency

2020 ◽  
Vol 16 (12) ◽  
pp. e1009055
Author(s):  
Zichong Li ◽  
Cyrus Hajian ◽  
Warner C. Greene
Keyword(s):  
Rna Polymerase Ii ◽  
Full Range ◽  
Screening Strategy ◽  
Host Factors ◽  
Host Cells ◽  
Hiv Latency ◽  
New Host ◽  
High Background ◽  
Hiv 1 ◽  
Host Genes

To counter HIV latency, it is important to develop a better understanding of the full range of host factors promoting latency. Their identification could suggest new strategies to reactivate latent proviruses and subsequently kill the host cells (“shock and kill”), or to permanently silence these latent proviruses (“block and lock”). We recently developed a screening strategy termed “Reiterative Enrichment and Authentication of CRISPRi Targets” (REACT) that can unambiguously identify host genes promoting HIV latency, even in the presence of high background “noise” produced by the stochastic nature of HIV reactivation. After applying this strategy in four cell lines displaying different levels of HIV inducibility, we identified FTSJ3, TMEM178A, NICN1 and the Integrator Complex as host genes promoting HIV latency. shRNA knockdown of these four repressive factors significantly enhances HIV expression in primary CD4 T cells, and active HIV infection is preferentially found in cells expressing lower levels of these four factors. Mechanistically, we found that downregulation of these newly identified host inhibitors stimulates different stages of RNA Polymerase II-mediated transcription of HIV-1. The identification and validation of these new host inhibitors provide insight into the novel mechanisms that maintain HIV latency even when cells are activated and undergo cell division.

scholarly journals Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

2001 ◽  
Vol 21 (17) ◽  
pp. 5767-5777 ◽  
Author(s):  
Amit Vas ◽  
Winnie Mok ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Safety Net ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
Origin Recognition ◽  
Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

scholarly journals BK Polyomavirus MicroRNA Levels in Exosomes Are Modulated by Non-Coding Control Region Activity and Down-Regulate Viral Replication When Delivered to Non-Infected Cells Prior to Infection

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 466 ◽  
Author(s):  
Francesco Martelli ◽  
Zongsong Wu ◽  
Serena Delbue ◽  
Fabian Weissbach ◽  
Maria Giulioli ◽  
...  
Keyword(s):  
Viral Replication ◽  
Point Mutations ◽  
T Antigen ◽  
Host Cells ◽  
Replication Capacity ◽  
Viral Gene ◽  
New Host ◽  
Infected Cells ◽  
Multiple Sclerosis Patients ◽  
Coding Control

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

Sign in / Sign up

Export Citation Format

Share Document