scholarly journals A novel DNA helicase with strand-annealing activity from the crenarchaeon Sulfolobus solfataricus

2007 ◽  
Vol 408 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Mariarita De Felice ◽  
Valentina Aria ◽  
Luca Esposito ◽  
Mariarosaria De Falco ◽  
Biagio Pucci ◽  
...  
Keyword(s):  
High Efficiency ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Sulfolobus Solfataricus ◽  
Genetic Material ◽  
Dna Helicase ◽  
Soluble Form ◽  
Monomeric Form ◽  
Genome Database ◽  
Strand Annealing

To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. Several proteins function at the interface of these DNA transactions. In the present study, we report on the identification of a novel archaeal DNA helicase. BlastP searches of the Sulfolobus solfataricus genome database allowed us to identify an open reading frame (SSO0112, 875 amino acid residues) having sequence similarity with the human RecQ5β. The corresponding protein, termed Hel112 by us, was produced in Escherichia coli in soluble form, purified to homogeneity and characterized. Gel-filtration chromatography and glycerol-gradient sedimentation analyses revealed that Hel112 forms monomers and dimers in solution. Biochemical characterization of the two oligomeric species revealed that only the monomeric form has an ATP-dependent 3′–5′ DNA-helicase activity, whereas, unexpectedly, both the monomeric and dimeric forms possess DNA strand-annealing capability. The Hel112 monomeric form is able to unwind forked and 3′-tailed DNA structures with high efficiency, whereas it is almost inactive on blunt-ended duplexes and bubble-containing molecules. This analysis reveals that S. solfataricus Hel112 shares some enzymatic features with the RecQ-like DNA helicases and suggests potential cellular functions of this protein.

scholarly journals The Short Life Span of Saccharomyces cerevisiae sgs1 and srs2 Mutants Is a Composite of Normal Aging Processes and Mitotic Arrest Due to Defective Recombination

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1531-1542 ◽  
Author(s):  
Mitch McVey ◽  
Matt Kaeberlein ◽  
Heidi A Tissenbaum ◽  
Leonard Guarente
Keyword(s):  
Cell Cycle ◽  
Life Span ◽  
Mitotic Cell ◽  
Dna Helicase ◽  
Premature Aging ◽  
Growth Defect ◽  
Short Life Span ◽  
Single Strand Annealing ◽  
Late Generation ◽  
Strand Annealing

Abstract Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.

scholarly journals Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

Archaea ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Giada Lo Gullo ◽  
Rosanna Mattossovich ◽  
Giuseppe Perugino ◽  
Anna La Teana ◽  
Paola Londei ◽  
...  
Keyword(s):  
High Efficiency ◽  
Specific Activity ◽  
Expression System ◽  
Sulfolobus Solfataricus ◽  
Essential Element ◽  
23S Rrna ◽  
Translation System ◽  
Cell Lysate ◽  
Cell Extracts

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins.

Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization

2019 ◽  
Vol 66 (6) ◽  
pp. 915-923
Author(s):  
Gokhan Gun ◽  
Rahmi Imamoglu ◽  
Ozge Tatli ◽  
Yuda Yurum ◽  
Ahmet Tarik Baykal ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Sulfolobus Solfataricus ◽  
Flavin Reductase

scholarly journals Soehngenia longivitae sp. nov., a Fermenting Bacterium Isolated from a Petroleum Reservoir in Azerbaijan, and Emended Description of the Genus Soehngenia

2020 ◽  
Vol 8 (12) ◽  
pp. 1967
Author(s):  
Tamara N. Nazina ◽  
Salimat K. Bidzhieva ◽  
Denis S. Grouzdev ◽  
Diyana S. Sokolova ◽  
Tatyana P. Tourova ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput Sequencing ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Phylogenomic Analysis ◽  
Rrna Gene ◽  
Petroleum Reservoir ◽  
Ph Range ◽  
The 16S Rrna Gene

A methanogenic enrichment growing on a medium with methanol was obtained from a petroleum reservoir (Republic of Azerbaijan) and stored for 33 years without transfers to fresh medium. High-throughput sequencing of the V4 region of the 16S rRNA gene revealed members of the genera Desulfovibrio, Soehngenia, Thermovirga, Petrimonas, Methanosarcina, and Methanomethylovorans. A novel gram-positive, rod-shaped, anaerobic fermentative bacterium, strain 1933PT, was isolated from this enrichment and characterized. The strain grew at 13–55 °C (optimum 35 °C), with 0–3.0% (w/v) NaCl (optimum 0–2.0%) and in the pH range of 6.7–8.0 (optimum pH 7.0). The 16S rRNA gene sequence similarity, the average nucleotide identity (ANI) and in silico DNA–DNA hybridization (dDDH) values between strain 1933PT and the type strain of the most closely related species Soehngenia saccharolytica DSM 12858T were 98.5%, 70.5%, and 22.6%, respectively, and were below the threshold accepted for species demarcation. Genome-based phylogenomic analysis and physiological and biochemical characterization of the strain 1933PT (VKM B-3382T = KCTC 15984T) confirmed its affiliation to a novel species of the genus Soehngenia, for which the name Soehngenia longivitae sp. nov. is proposed. Genome analysis suggests that the new strain has potential in the degradation of proteinaceous components.

scholarly journals Identification of Lead Compounds Targeting the Cathepsin B-Like Enzyme of Eimeria tenella

2011 ◽  
Vol 56 (3) ◽  
pp. 1190-1201 ◽  
Author(s):  
Marie Schaeffer ◽  
Joerg Schroeder ◽  
Anja R. Heckeroth ◽  
Sandra Noack ◽  
Michael Gassel ◽  
...  
Keyword(s):  
Cathepsin B ◽  
Biochemical Characterization ◽  
Single Copy ◽  
Eimeria Tenella ◽  
Genome Database ◽  
Content Type ◽  
Cysteine Peptidase ◽  
Peptidase Inhibitor ◽  
Mature Enzyme ◽  
Novel Drug

ABSTRACTCysteine peptidases have been implicated in the development and pathogenesis ofEimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database ofEimeria tenella(EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme fromPichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery againstEimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.

scholarly journals Genetic and Biochemical Characterization of the Streptococcus pneumoniae PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

2006 ◽  
Vol 188 (21) ◽  
pp. 7416-7425 ◽  
Author(s):  
J. A. Ruiz-Masó ◽  
S. P. Anand ◽  
M. Espinosa ◽  
S. A. Khan ◽  
G. del Solar
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biochemical Characterization ◽  
Essential Gene ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Rolling Circle Replication ◽  
Lower Efficiency ◽  
Rolling Circle ◽  
Rep Protein

ABSTRACT PcrA is a chromosomally encoded DNA helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. Efficient interaction between PcrA and the plasmid-encoded replication initiator (Rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a Rep protein to interact with heterologous PcrA helicases has been invoked as a determinant of plasmid promiscuity. We characterized transcription of the Streptococcus pneumoniae pcrA gene in its genetic context and studied the biochemical properties of its product, the PcrA Spn helicase. Transcription of the pneumococcal pcrA gene was directed by promoter Pa, consisting of an extended −10 box. Promoter Pa also accounted for expression of a second essential gene, radC, which was transcribed with much lower efficiency than pcrA, probably due to the presence of a terminator/attenuator sequence located between the two genes. PcrA Spn displayed single-stranded DNA-dependent ATPase activity. PcrA Spn showed 5′→3′ and 3′→5′ helicase activities and bound efficiently to partially duplex DNA containing a hairpin structure adjacent to a 6-nucleotide 5′ or 3′ single-stranded tail and one unpaired (flap) nucleotide in the complementary strand. PcrA Spn interacted specifically with RepC, the initiator of staphylococcal plasmid pT181. Although the pneumococcal helicase was able to initiate unwinding of the RepC-nicked pT181 DNA, it was much less processive in this activity than the cognate staphylococcal PcrA protein. Accordingly, PcrA Spn was inefficient in in vitro replication of pT181, and perhaps as a consequence, this plasmid could not be established in S. pneumoniae.

scholarly journals Requirement of RAD52 Group Genes for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (21) ◽  
pp. 7758-7764 ◽  
Author(s):  
Venkateswarlu Gangavarapu ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Template Switching ◽  
Template Strand ◽  
Fork Regression ◽  
Strand Annealing ◽  
Almost All ◽  
Annealing Mode ◽  
Damaged Dna

ABSTRACT In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases η and ζ and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.

scholarly journals Response of the Hyperthermophilic Archaeon Sulfolobus solfataricus to UV Damage

2007 ◽  
Vol 189 (23) ◽  
pp. 8708-8718 ◽  
Author(s):  
Sabrina Fröls ◽  
Paul M. K. Gordon ◽  
Mayi Arcellana Panlilio ◽  
Iain G. Duggin ◽  
Stephen D. Bell ◽  
...  
Keyword(s):  
Dna Microarrays ◽  
Sulfolobus Solfataricus ◽  
Genetic Material ◽  
Transcriptional Response ◽  
Microscopic Analysis ◽  
Large Cell ◽  
Recombinational Repair ◽  
Supporting Evidence ◽  
Uv Damage ◽  
Dna Double Strand Breaks

ABSTRACT In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.

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