Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1

Berkner, Silvia; Hinojosa, Mery Pina; Prangishvili, David; Lipps, Georg

doi:10.1002/mbo3.198

Cited by 7 publications

(6 citation statements)

References 39 publications

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“…For cloning of pSVAmZ-SH10 the minimal replicon of pRN1 consisting of the region surrounding orf56 and orf904 was shortened and the remaining NdeI site simultaneously removed 47 . To this end, two backbone fragments were amplified using the primer combinations mod_Size_fw, mod_NdeI_del_rev and mod_NdeI_del_fw, mod_Size_rev and assembled by overlap PCR.…”

Section: Methodsmentioning

confidence: 99%

Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

et al. 2017

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Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies. Here, we demonstrate that in vivo activity-based protein profiling (ABPP) may be used to sensitively detect either native or heterogeneously expressed active enzymes in living archaea even under these extreme conditions. In combination with the development of a genetically engineered archaeal screening strain, ABPP can furthermore be used in functional enzyme screenings from (meta)genome samples. We anticipate that our ABPP approach may therefore find application in basic archaeal research but also in the discovery of novel enzymes from (meta)genome libraries.

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Section: Methodsmentioning

confidence: 99%

Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

et al. 2017

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“…All family members share a conserved genomic region and three highly conserved gene products which have been characterised biochemically (Peng et al, 2000). Recent studies have revealed that a single multifunctional replication protein (ORF904) with helicase, primase and DNA polymerase activity, reminiscent of the type of replication performed by some linear bacteriophages, is required to initiate plasmid replication (Lipps, 2011;Berkner et al, 2014 marked contrast to all DNA polymerases, ORF904 is able to polymerise DNA de novo, i.e. without a primer.…”

Section: Small Cryptic Plasmidsmentioning

confidence: 99%

The Flexible Genome of Acidophilic Prokaryotes

Beard¹,

Ossandon²,

Rawlings³

et al. 2021

Current Issues in Molecular Biology

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Over the last couple of decades there has been considerable progress in the identification and understanding of the mobile genetic elements that are exchanged between microbes in extremely acidic environments, and of the genes piggybacking on them. Numerous plasmid families, unique viruses of bizarre morphologies and lyfe cycles, as well as plasmid-virus chimeras, have been isolated from acidophiles and characterized to varying degrees. Growing evidence provided by omic-studies have shown that the mobile elements repertoire is not restricted to plasmids and viruses, but that a plethora of integrative elements ranging from miniature inverted repeat transposable elements to large integrative conjugative elements populate the genomes of acidophilic bacteria and archaea. This article reviews the diversity of elements that have been found to constitute the flexible genome of acidophiles. Special emphasis is put on the knowledge generated for Sulfolobus (archaea) and species of the bacterial genera Acidithiobacillus and Leptospirillum. Also, recent knowledge on the strategies used by acidophiles to contain deletereous exchanges while allowing innovation, and the emerging details of the molecular biology of these systems, are discussed. Major lacunae in our understanding of the mobilome of acidophilic prokaryotes and topics for further investigations are identified.

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“…However, unlike conventional AEP members that function as replication initiation-specific primases, PrimPols play diverse roles in different organisms (Guilliam et al, 2015). For example, PrimPol from archaeal plasmid pRN1 (ORF904) is suggested to be a replicative DNA polymerase (Berkner et al, 2014), while human PrimPol (hPrimPol) is involved in DNA damage tolerance (Bianchi et al, 2013;García-Gómez et al, 2013). Recently, it is reported that a PrimPol encoded in a mobile element of Thermus thermophilus is contributed in defense against invading DNA (García-Quintans et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase

Huang

et al. 2021

Front. Microbiol.

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PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA de novo.

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Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1

Cited by 7 publications

References 39 publications

Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

The Flexible Genome of Acidophilic Prokaryotes

Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase

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