pIT3, a cryptic plasmid isolated from the hyperthermophilic crenarchaeon Sulfolobus solfataricus IT3

Prato, Santina; Cannio, Raffaele; Klenk, Hans‐Peter; Contursi, Patrizia; Rossi, Mosè; Bartolucci, Simonetta

doi:10.1016/j.plasmid.2006.02.002

Cited by 15 publications

(17 citation statements)

References 43 publications

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“…It is a member of the pRN family of genetic elements, comprising pRN1, pRN2, pDL10, pHEN7, and pSSVx (3,22,23,25,34). The more recently described plasmids pTIK4, pTAU4, pORA1 (18), and pIT3 (35) also contain open reading frames with sequence similarity to open reading frames from the pRN family plasmids. The sequence of pRN1 has been determined (22), and six open reading frames have been identified.…”

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confidence: 99%

Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 fromSulfolobus islandicusREN1H1 and Regulation of Its Replication Operon

Berkner

Lipps

2007

J Bacteriol

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The plasmid pRN1 from Sulfolobus islandicus REN1H1 belongs to the crenarchaeal plasmid family pRN. The plasmids in this family encode three conserved proteins that participate in plasmid replication and copy number regulation, as suggested by biochemical characterization of the recombinant proteins. In order to deepen our understanding of the molecular biology of these plasmids, we investigated the transcriptional activity of the model plasmid pRN1. We detected five major transcripts present at about 2 to 15 copies per cell. One long transcriptional unit comprises the genes for the plasmid-copy-number control protein Orf56/CopG and the replication protein Orf904. A second transcript with a long 3-untranslated region codes for the DNA binding protein Orf80. For both transcripts, we identified countertranscripts which could play a regulatory role. The function of the fifth transcript is unclear. For the five transcripts, we determined the start site, the transcript end, the stability, and the abundance in different growth phases. Reporter gene experiments demonstrated that the copy number control protein Orf56 represses transcription of the orf56-orf904 cotranscript in vivo.The plasmid pRN1 (5,350 bp) has been isolated from Sulfolobus islandicus strain REN1H1 (47) and occurs natively together with plasmid pRN2 in its host strain but has been shown to replicate independently of pRN2 (36). It is a member of the pRN family of genetic elements, comprising pRN1, pRN2, pDL10, pHEN7, and pSSVx (3,22,23,25,34). The more recently described plasmids pTIK4, pTAU4, pORA1 (18), and pIT3 (35) also contain open reading frames with sequence similarity to open reading frames from the pRN family plasmids.

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confidence: 99%

Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 fromSulfolobus islandicusREN1H1 and Regulation of Its Replication Operon

Berkner

Lipps

2007

J Bacteriol

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“…The N-terminal domain comprising residues 31-245 of the orf915-encoded putative replication protein of the plasmid pIT3 was predicted to be the minimum-length sequence containing all the functionally relevant structural motifs [18]. This domain (without the 30 N-terminal residues) was modeled onto the crystallographic structure of the orf904-encoded bifunctional prim-pol domain of the archaeal plasmid pRN1 (PDB entry 1RN1) [13], which following PSI-BLAST sequence search against PDB and FUGUE server fold recognition was found to be the best possible structural template.…”

Section: Homology Modeling and Structure-sequence Analysismentioning

confidence: 99%

“…2A). As described in the Experimental Procedures, the deletion gene was amplified using the PCR of the S. solfataricus plasmid pIT3 [18] and then cloned into pET-30c(+). In E. coli, the recombinant protein (from now on Rep245) was highly overexpressed as a fusion with the C-terminal six-residue histidine tail (LEHHHHHH).…”

Section: Expression and Protein Purificationmentioning

confidence: 99%

“…In a previous study, we reported the findings of an analysis of the complete sequence of the cryptic plasmid pIT3 isolated from the crenarchaeon S. solfataricus strain IT3 [18]. The fully sequenced plasmid contains six ORFs, the largest of which (ORF915) spans over half the plasmid genome and encodes a putative 100 kDa replication protein designated as RepA [18].…”

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confidence: 99%

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Molecular modeling and functional characterization of the monomeric primase–polymerase domain from the Sulfolobus solfataricus plasmid pIT3

et al. 2008

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In all cell types, chromosomal DNA replication is a complex process entailing three enzymatic activities: helicase activity for double-helix unzipping and primase and DNA polymerase for RNA primer de novo synthesizing and elongation respectively [1,2].Based on the biochemical data accumulated to date, archaeal DNA replication involves a smaller number of polypeptides at each stage of the process and is thus just a simpler form of the much more complex eukaryotic replication machinery [3][4][5][6]. Nonetheless, Archaea are not simply 'mini Eukarya'. A better definition would be 'a mosaic of eukaryal and bacterial systems with specific archaeal features'. Aspects worth mentioning in this respect are the promiscuous nature of the nucleic acid functions performed by archaeal primases and the dual, template-dependent and A tri-functional monomeric primase-polymerase domain encoded by the plasmid pIT3 from Sulfolobus solfataricus strain IT3 was identified using a structural-functional approach. The N-terminal domain of the pIT3 replication protein encompassing residues 31-245 (i.e. Rep245) was modeled onto the crystallographic structure of the bifunctional primase-polymerase domain of the archaeal plasmid pRN1 and refined by molecular dynamics in solution. The Rep245 protein was purified following overexpression in Escherichia coli and its nucleic acid synthesis activity was characterized. The biochemical properties of the polymerase activity such as pH, temperature optima and divalent cation metal dependence were described. Rep245 was capable of utilizing both ribonucleotides and deoxyribonucleotides for de novo primer synthesis and it synthesized DNA products up to several kb in length in a template-dependent manner. Interestingly, the Rep245 primase-polymerase domain harbors also a terminal nucleotidyl transferase activity, being able to elongate the 3¢-end of synthetic oligonucleotides in a non-templated manner. Comparative sequence-structural analysis of the modeled Rep245 domain with other archaeal primase-polymerases revealed some distinctive features that could account for the multifaceted activities exhibited by this domain. To the best of our knowledge, Rep245 typifies the shortest functional domain from a crenarchaeal plasmid endowed with DNA and RNA synthesis and terminal transferase activity.

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“…Plasmid pIT3 (39), isolated from a Sulfolobus solfataricus strain from Italy, and three plasmids, pTAU4, pORA1, and pTIK4 (59), obtained from Sulfolobus neozealandicus from New Zealand, encode replication proteins different from ORF904. The replication proteins from pORA1 and pIT3 harbor the N-terminal prim-pol domain (16,40).…”

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A Superfamily 3 DNA Helicase Encoded by Plasmid pSSVi from the Hyperthermophilic ArchaeonSulfolobus solfataricusUnwinds DNA as a Higher-Order Oligomer and Interacts with Host Primase

Guo

Huang

2010

J Bacteriol

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Replication proteins encoded by nonconjugative plasmids from the hyperthermophilic archaea of the order Sulfolobales show great diversity in amino acid sequence. We have biochemically characterized ORF735, a replication protein from pSSVi, an integrative nonconjugative plasmid from Sulfolobus solfataricus P2. We show that ORF735 is a DNA helicase of superfamily 3. It unwound double-stranded DNA (dsDNA) in a 3-to-5 direction in the presence of ATP over a wide range of temperatures, from 37°C to 75°C, and possessed DNA-stimulated ATPase activity. ORF735 existed in solution as a salt-stable dimer and was capable of assembling into a salt-sensitive oligomer that was significantly larger than a hexamer in the presence of a divalent cation (Mg 2؉ ) and an adenine nucleotide (ATP, dATP, or ADP) or its analog (ATP␥S or AMPPNP). Both N-terminal and C-terminal portions of ORF735 (87 and 160 amino acid residues, respectively, in size) were required for protein dimerization but dispensable for the formation of the higher-order oligomer. The protein unwound DNA only as a large oligomer. Yeast two-hybrid and coimmunoprecipitation assays revealed that ORF735 interacted with the noncatalytic subunit of host primase. These findings provide clues to the functional role of ORF735 in pSSVi DNA replication.DNA helicases are ubiquitous motor proteins that utilize the energy of nucleotide triphosphate hydrolysis to translocate along and unwind the duplex DNA in DNA replication, transcription, recombination, and repair (30). These enzymes often exist as oligomers such as hexamers or dimers. Hexameric DNA helicases are widely found in cellular forms of life as well as viruses and plasmids (13,44,45,48,54). Both bacterial helicases (represented by Escherichia coli DnaB) (5) and eukaryotic helicases (represented by mammalian MCM) (21, 57) have been extensively studied. Electron microscopy (EM) and image studies have revealed a common ring-shaped structure for all known hexameric helicases (36). This ring-like structure allows the enzyme to encircle the DNA and translocate in a processive fashion.Most extrachromosomal genetic elements encode their own DNA helicases (13,44,45,48,54). Our knowledge of these helicases has been derived primarily from studies of bacterial and eukaryotic viruses and plasmids. These genetic elements are replicated through the concerted action of self-encoded and recruited host replication proteins. Many viral or plasmid helicases serve more functions than DNA unwinding. For example, the simian virus 40 (SV40) large T antigen is responsible for the recognition as well as the unwinding of the viral replication origin (9-11). The bifunctional T7 gp4 protein possesses both helicase and primase activities, and its C-terminal helicase domain interacts with T7 DNA polymerase to coordinate helicase and polymerase activities (34).Replicative helicases from the archaea, the third domain of life, have attracted much attention in the past decade. Since the first report of the biochemical properties of an archaeal MCM protein...

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pIT3, a cryptic plasmid isolated from the hyperthermophilic crenarchaeon Sulfolobus solfataricus IT3

Cited by 15 publications

References 43 publications

Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 fromSulfolobus islandicusREN1H1 and Regulation of Its Replication Operon

Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 fromSulfolobus islandicusREN1H1 and Regulation of Its Replication Operon

Molecular modeling and functional characterization of the monomeric primase–polymerase domain from the Sulfolobus solfataricus plasmid pIT3

A Superfamily 3 DNA Helicase Encoded by Plasmid pSSVi from the Hyperthermophilic ArchaeonSulfolobus solfataricusUnwinds DNA as a Higher-Order Oligomer and Interacts with Host Primase

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