(none).
Identification of essential and non-essential single-stranded DNA-binding proteins in a model archaeal organism.
Skowyra A, MacNeill SA
Nucleic Acids Res. 40(3):1077-90 (2012).
HVO_0291, HVO_1337, MA0591, MA3018
HQ_1435A, HVO_0292, HVO_0519, HVO_1338, RPA2, RPA3, Rpa, replication protein A
HVO_1336, Rpe, rpe
HQ_1435A, HVO_0292, HVO_0519, HVO_1338, RPA2, RPA3, Rpa, replication protein A
endA
...Abstract:
Identification of essential and non-essential single-stranded DNA-binding proteins in a model archaeal organism Agnieszka Skowyra and Stuart A. MacNeill* School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9TF, UK ABSTRACT Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies. INTRODUCTION Single-stranded DNA-binding proteins (SSBs) are indispensable for many aspects of DNA metabolism including replication, repair and recombination, and play a vital role in the maintenance of genomic stability in all three domains of life (1,2). SSBs are characterized by the presence of one or more OB (oligosaccharide oligonucleotide binding) fold domains. OB folds consist of a ?ve-stranded b-sheet that is coiled to form a closed b-barrel, often capped by an a-helix (2,3). They range in length from 75 1...
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Genetic and Biochemical Identification of a Novel Single-Stranded DNA-Binding Complex in Haloferax volcanii.
Stroud A, Liddell S, Allers T
Front Microbiol. 3:224 (2012).
RPA-associated protein, RPAP1, RPAP3, rpa-associated protein, rpap1, rpap3
RPA1, RPA2, RPA3, Replication factor-A, replication protein A, rpa, rpa1, rpa2, rpa3
rpe
RPA1, RPA2, RPA3, Replication factor-A, replication protein A, rpa, rpa1, rpa2, rpa3
...Abstract:Amy Stroud 1, Susan Liddell 2 and Thorsten Allers 1 * School of Biology, Queen s Medical Centre, University of Nottingham, Nottingham, UK Division of Animal Sciences, University of Nottingham, Loughborough, UK Edited by: Zvi Kelman, University of Maryland, USA Reviewed by: Jocelyne DiRuggiero, The Johns Hopkins University, USA Stuart MacNeill, University of St Andrews, UK *Correspondence: Thorsten Allers, School of Biology, Queen s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK. e-mail: thorsten.allers@ nottingham.ac.uk Single-stranded DNA (ssDNA)-binding proteins play an essential role in DNA replication and repair. They use oligonucleotide/oligosaccharide-binding (OB)-folds, a ?ve-stranded ?-sheet coiled into a closed barrel, to bind to ssDNA thereby protecting and stabilizing the DNA. In eukaryotes the ssDNA-binding protein (SSB) is known as
replication protein A (
/) (RPA) and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the
rpa (/) genes (
rpa1 (/) and
rpa3 (/)) exist in operons with a novel gene speci?c to Euryarchaeota; this gene encodes a protein that we have termed
RPA-associated protein () (rpap). The rpap genes encode proteins belonging to COG3390 group and feature OB-folds, suggesting that they might cooperate with RPA in binding to ssDNA. Our genetic analysis showed that
rpa1 (/) and
rpa3 (/) deletion mutants have differing phenotypes; only ?rpa3 strains are hypersensitive to DNA damaging agents. Deletion of the
rpa3 (/)-associated gene
rpap3 () led to similar levels of DNA damage sensitivity, as did deletion of the
rpa3 (/) operon, suggesting that
RPA3 (/) and
RPAP3 () function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that
RPA3 (/) co-puri?es with
RPAP3 (), and
RPA1 (/) co-puri?es with RPAP1.This indicates that the RPAs interact only with their respective as...
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Histone and TK0471/TrmBL2 form a novel heterogeneous genome architecture in the hyperthermophilic archaeon Thermococcus kodakarensis.
Maruyama H, Shin M, Oda T, Matsumi R, Ohniwa RL, Itoh T, Shirahige K, Imanaka T, Atomi H, Yoshimura SH, Takeyasu K
Mol Biol Cell. 22(3):386-98 (2011).
Abstract:
Hugo Maruyamaa, Minsang Shina, Toshiyuki Odaa, Rie Matsumib, Ryosuke L. Ohniwac, Takehiko Itohd, Katsuhiko Shirahigee, Tadayuki Imanakaf, Haruyuki Atomig, Shige H. Yoshimuraa, and Kunio Takeyasua a Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan; bLaboratory of Microbiology, Wageningen University, 6703 HB Wageningen, The Netherlands; c Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8575, Japan; dLaboratory of In Silico Functional Genomics, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan; eLaboratory of Genome Structure and Function, Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan; fDepartment of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu 525-8577, Japan; gDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan ABSTRACT Being distinct from bacteria and eukaryotes, Archaea constitute a third domain of living things. The DNA replication, transcription, and translation machineries of Archaea are more similar to those of eukaryotes, whereas the genes involved in metabolic processes show more similarity to their bacterial counterparts. We report here that TK0471/TrmB-like 2 (TrmBL2), in addition to histone, is a novel type of abundant chromosomal protein in the model euryarchaeon Thermococcus kodakarensis. The chromosome of T. kodakarensis can be separated into regions enriched either with histone, in which the genetic material takes on a ?beads-on-a-string? appearance, or with TK0471/TrmBL2, in which it assumes a thick ?brous structure. TK0471/TrmBL2 binds to both coding and intergenic regions and represses transcription when bound to the promoter region. These results show tha...
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Affinity purification of an archaeal DNA replication protein network.
Li Z, Santangelo TJ, Cubonova L, Reeve JN, Kelman Z
MBio. 1(5) (2010).
TK1959
Replication protein A, TK1961
Replication protein A, TK1961
...Abstract:
Information about commercial reprint orders: http://mbio.asm.org/misc/reprints.xhtml Information about Print on Demand and other content delivery options: http://mbio.asm.org/misc/contentdelivery.xhtml To subscribe to another ASM Journal go to: http://journals.asm.org/subscriptions/ Af?nity Puri?cation of an Archaeal DNA Replication Protein Network Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA, and Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA,a and Department of Microbiology, Ohio State University, Columbus, Ohio, USAb ABSTRACT Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His6-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His6-tagged proteins, stable complexes assembled in vivo have been isolated directly from clari?ed cell lysates and the T. kodakarensis proteins present have been identi?ed by mass spectrometry. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that con?rms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a ?rm experimental foundation for archaeal replication research. The proteins identi?ed and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts. IMPORTANCE DNA replication is a central and essential event in all cell cycles. Historically, the biological world was divided into prokaryotes and eukaryotes, based on the absence or presence of a nuclear membrane, and many components of the DNA replication machinery have been identi?ed and characterized as conserved or nonconserved in prokaryotic versus eukaryotic organisms. However, it is now known that there ar...
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Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination.
Komori K, Ishino Y
J Biol Chem. 276(28):25654-60 (2001).
AF0779, Ta0388, Vng1253c, Vng2162c
AF0382, AF0780, Ta0387, Ta1149, Vng0133 g, Vng1255c, Vng2160c
AF0382, AF0780, Ta0387, Ta1149, Vng0133 g, Vng1255c, Vng2160c
...Abstract:
Kayoko Komori and Yoshizumi Ishino? From the Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus. Single-stranded DNA-binding protein (SSB)1 in Bacteria and replication protein A (RPA) in Eukarya play essential roles in DNA replication, recombination, and repair. (1 3). The bacterial SSB and the eukaryotic RPA bind to single-stranded DNA as a homotetramer and a heterotrimer, respectively. Although there is little amino acid sequence similarity between SSB and RPA, recent analyses of the three-...
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