The sub-cellular localization of 
            Sulfolobus
             DNA replication

Gristwood, Tamzin; Duggin, Iain G.; Wagner, Michaela; Albers, Sonja‐Verena; Bell, Stephen D.

doi:10.1093/nar/gks217

Cited by 29 publications

(33 citation statements)

References 37 publications

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“…In uninfected cells, PCNA showed a peripheral multifocus distribution (Fig. 2, 0 hpi), as described earlier for Sulfolobus acidocaldarius (21). In contrast, PCNA is redistributed to a single focus overlapping with that of gp17 in SIRV2-infected cells ( Next, we examined the intracellular distribution of host DNA polymerases which are implicated in SIRV2 replication because the viral genome does not encode a DNA polymerase (6).…”

Section: Resultssupporting

confidence: 57%

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Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

Martínez-Álvarez

Deng

Peng

2017

J Virol

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Viral factories are compartmentalized centers for viral replication and assembly in infected eukaryotic cells. Here, we report the formation of a replication focus by prototypical archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in the model archaeon Sulfolobus. This rod-shaped virus belongs to the viral family Rudiviridae, carrying linear double-stranded DNA (dsDNA) genomes, which are very common in geothermal environments. We demonstrate that SIRV2 DNA synthesis is confined to a focus near the periphery of infected cells. Moreover, viral and cellular replication proteins are recruited to, and concentrated in, the viral replication focus. Furthermore, we show that of the four host DNA polymerases (DNA polymerase I [Dpo1] to Dpo4), only Dpo1 participates in viral DNA synthesis. This constitutes the first report of the formation of a viral replication focus in archaeal cells, suggesting that organization of viral replication in foci is a widespread strategy employed by viruses of the three domains of life. IMPORTANCEThe organization of viral replication in foci or viral factories has been mostly described for different eukaryotic viruses and for several bacteriophages. This work constitutes the first report of the formation of a viral replication center by a virus infecting members of the Archaea domain.KEYWORDS replication focus, archaeal virus, SIRV2, DNA polymerase T he compartmentalization of viral genome replication is a well-known feature of many eukaryotic viruses, where replication is confined to specific subcellular microenvironments termed viral factories, viroplasms, viral replication compartments, or viral replication centers. In general, viral factories function as a scaffold containing viral genomes and proteins involved in viral replication and assembly, and this is hypothesized to increase the efficiency of viral replication and confer protection from the cellular antiviral immune responses (1, 2).The formation of viral factories has been observed for DNA, double-stranded RNA (dsRNA), positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA viruses, and it has been extensively studied for nucleocytoplasmic large DNA viruses (e.g., poxviruses and asfarviruses) and positive-sense ssRNA viruses, including flaviviruses, coronaviruses, picornaviruses, and togaviruses (1). Although these viruses infect a wide range of hosts and their corresponding viral factories differ in their morphologies, components, and intracellular locations, their modes of formation share some similarities. First, formation of viral factories usually involves extensive organizational changes in the host cell membranes and/or cytoskeleton. Second, viral factories constitute a scaffold for the concentration of viral genomes and of viral and cellular proteins required for viral replication and assembly (1, 2).To date, the formation of viral factories has been studied mainly for eukaryotic viruses, although the organization of viral replication has been reported for bacteriophages 29, PRD1, SSP1, and 20...

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Section: Resultssupporting

confidence: 57%

“…Interestingly, PCNA distribution in Sulfolobus cells, in the absence of viral infection, is also peripheral (21). Therefore, attachment to the membrane could be a cellular mechanism for organizing replisomes that the virus exploits to its own advantage.…”

Section: Discussionmentioning

confidence: 99%

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

Martínez-Álvarez

Deng

Peng

2017

J Virol

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“…S. acidocaldarius SacTK, a pyrEF − TK + strain derived from S. acidocaldarius DSM639 (28), was grown at 75°C in Brock's medium at pH 3.2 containing 0.1% (wt/vol) tryptone, 0.2% (wt/vol) xylose, and 0.025 mg/mL uracil. After transformations, SacTK cultures contained 0.1% NZ-amine as an alternative to tryptone.…”

Section: Methodsmentioning

confidence: 99%

“…The construct for tagging S. acidocaldarius cdc45 was generated by overlap PCR (Table S1) and transformation of S. acidocaldarius strain SacTK (28). Cdc45 was overexpressed in S. islandicus E223S, using the pSSR vector (23).…”

Section: Methodsmentioning

confidence: 99%

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

Gristwood²,

Hodgson³

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS • Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome.T he initiation of DNA replication is an important control point in the progression of the cell cycle. In eukaryotes, origins are defined by the initiator protein ORC complex that, via the actions of two additional factors, the helicase coloaders Cdc6 and Cdt1, directs loading of the MCM(2-7) replicative helicase onto doublestranded DNA. Activation of the MCM(2-7) replicative helicase occurs subsequent to recruitment, leading to DNA melting and assembly of the full replisome apparatus (1, 2). Key steps in MCM activation involve a series of phosphorylation-dependent events that promote the sequential association of Cdc45 and the GINS complex with the chromatin-associated MCM double hexamer. These recruitment events are regulated by the CDK and DDK kinases and require the additional accessory factors Sld3/7, Dpb11, and Sld2(3-6). The Cdc45•MCM(2-7)•GINS complex (CMG) forms the core of the eukaryotic replisome, and this 11-subunit assembly appears to be the functional helicase driving fork progression (3-6). The archaeal replication machinery resembles an ancestral form of its eukaryotic counterpart. Archaea possess a simple homohexameric MCM (5, 7). In addition, archaeal homologs of GINS and Cdc45 have been identified (8)(9)(10)(11)(12)(13)(14). In species of the genus Sulfolobus, we have previously demonstrated that the GINS complex interacts with the N-terminal domains of MCM (8). In Sulfolobus, GINS is a dimer of dimers: one subunit, Gins23, is related to the eukaryotic GINS components Psf2 and Psf3, and the second Sulfolobus subunit, Gins15, is related to the eukaryotic Sld5 and Psf1. These sequence relationships have been confirmed by structural studies of the Thermococcus kodakarensis GINS complex that have demonstrated the tetrameric assembly of archaeal (Gins15) 2 • (Gins23) 2 and validated the organizational similarity of the archaeal and eukaryotic GINS complexes (15). Interestingly, Sulfolobus GINS copurifies over the course of eight steps with a further polypeptide that we initially named RecJdbh, based on its observed homology with the presumptive DNA binding domain of the bacterial exonuclease, RecJ (8). Subsequent sequence analyses have revealed a relationship between RecJ and eukaryotic Cdc45, and this has been elegantly confirmed by recent structural studies of eukaryotic Cdc45 (9,11,16,17). We therefore propose renaming RecJdbh as Cdc45. As archaea lack orthologs of Sld2, Sld...

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“…Revealing and subcellu lar localization of DNA replication, but already with the help of EdU, in the Schizosaccharomyces pombe were also coupled with a necessity to construct the strain expressing TK gene of the herpes virus and hENT1 gene encoding the nucleoside transporter. For hyperthermophilic archaea Sulfolobus sp., the appli cation of EdU for the monitoring of DNA replication became possible after the construction of strains expressing TK from another species of hyperthermo philic archaea [17,18].…”

Section: Results and Discussion The Presence Of Thymidine Kinase Detementioning

confidence: 99%

Extra perspectives of 5-ethynyl-2′-deoxyuridine click reaction with fluorochrome azides to study cell cycle and deoxyribonucleoside metabolism

Носов

Фоменков

Mamaeva

et al. 2014

Russ J Plant Physiol

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Beginning with the pioneering work of Salic and Mitchison (2008), the application of thymidine analogue 5 ethynyl 2' deoxyuridine (EdU) for the detection of cells replicating DNA is actively expanding. Being incorporated into DNA, this nucleoside after click reaction of azide-alkyne cycloaddition with azides of fluorochromes can be easily detected by fluorescence. Recently, protocols of EdU application in combi nation with click reaction adapted for plant cells appeared, and they are help for a monitoring S period of the cell cycle in the root meristems and in vitro cultured cells with the help of a microscope and flow cytometer. In this work, we focused some details of developed methods and their modifications and also recommended new protocols. In particular, we suggested combining EdU incorporation into the cells replicating DNA with subsequent isolation of protoplasts from them and their preparation for the microscopic analysis and flow cytometry. In addition, the method of determination of EdU phosphorylation dynamics in the cells in vivo is suggested.Abbreviations: 7 AAD-7 aminoactinomycin D; BrdU-5 bromo 2' deoxyuridine; CC-cell cycle; DAPI-4',6 diami dino 2 phenylindol; DMSO-dimethylsulfoxide; dT-thymi dine; EdU-5 ethynyl 2' deoxyuridine; MMC-mithramycin A; PBS-phosphate buffered saline; PI-propidium iodide; TK-thymidine kinase; TPFC-two parameter flow cytometry.

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The sub-cellular localization of Sulfolobus DNA replication

Cited by 29 publications

References 37 publications

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

Extra perspectives of 5-ethynyl-2′-deoxyuridine click reaction with fluorochrome azides to study cell cycle and deoxyribonucleoside metabolism

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