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Expression, purification, crystallization and preliminary X-ray analysis of the RecQ helicase catalytic core from Deinococcus radiodurans.

ABSTRACT: The RecQ proteins are a highly conserved group of DNA helicases which play crucial roles in the maintenance of genome stability. DrRecQ from the radioresistant bacterium Deinococcus radiodurans is a special member of the RecQ family because it contains three Helicase-and-RNase-D-C-terminal (HRDC) domains at the C-terminus. The helicase catalytic core is essential for ATPase and DNA-unwinding activities. In this work, the helicase catalytic core of DrRecQ was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour diffusion method and X-ray diffraction data were collected to 2.9?Å resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.75, b = 95.61, c = 183.83?Å.

SUBMITTER: Chen SC

PROVIDER: S-EPMC3490466 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Expression, purification, crystallization and preliminary X-ray analysis of the RecQ helicase catalytic core from Deinococcus radiodurans.

Chen Sheng-Chia SC Huang Chi-Hung CH Yang Chia-Shin CS Chang Chi-Huang CH Kuan Shu-Min SM Chan Nei-Li NL Chen Yeh Y

Acta crystallographica. Section F, Structural biology and crystallization communications 20120929 Pt 10

The RecQ proteins are a highly conserved group of DNA helicases which play crucial roles in the maintenance of genome stability. DrRecQ from the radioresistant bacterium Deinococcus radiodurans is a special member of the RecQ family because it contains three Helicase-and-RNase-D-C-terminal (HRDC) domains at the C-terminus. The helicase catalytic core is essential for ATPase and DNA-unwinding activities. In this work, the helicase catalytic core of DrRecQ was expressed in Escherichia coli, purifi ...[more]

PMID: 23027755

Similar Datasets

Crystal structure of Deinococcus radiodurans RecQ helicase catalytic core domain: the interdomain flexibility.

Project description:RecQ DNA helicases are key enzymes in the maintenance of genome integrity, and they have functions in DNA replication, recombination, and repair. In contrast to most RecQs, RecQ from Deinococcus radiodurans (DrRecQ) possesses an unusual domain architecture that is crucial for its remarkable ability to repair DNA. Here, we determined the crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form, revealing interdomain flexibility in its first RecA-like and winged-helix (WH) domains. Additionally, the WH domain of DrRecQ is positioned in a different orientation from that of the E. coli RecQ (EcRecQ). These results suggest that the orientation of the protein during DNA-binding is significantly different when comparing DrRecQ and EcRecQ.

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Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase.

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Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans.

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Purification, crystallization and preliminary X-ray crystallographic analysis of branched-chain aminotransferase from Deinococcus radiodurans.

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Purification, crystallization and preliminary X-ray diffraction analysis of DNA damage response A protein from Deinococcus radiodurans.

Project description:DNA damage response A protein (DdrA) from Deinococcus radiodurans has been suggested to be involved in DNA-repair processes through binding to 3'-ends of single-stranded DNA, thereby protecting the ends from nuclease digestion. In this study, a recombinant C-terminally truncated form of D. radiodurans DdrA (DdrA157) comprising the first 157 residues of DdrA was expressed in Escherichia coli, purified and crystallized. Single crystals of DdrA157 were obtained by the hanging-drop method at 293?K. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a=46.31, b=180.26, c=114.17?Å, ?=90.02°. The crystal was expected to contain 14 molecules in the asymmetric unit. Diffraction data were collected to 2.35?Å resolution on beamline BL-5 at Photon Factory and initial phase determinations were attempted by the molecular-replacement method using the human Rad52 structure.

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Expression, purification and preliminary X-ray crystallographic analysis of UDP-galactopyranose mutase from Deinococcus radiodurans.

Project description:UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. A UGM-substrate complex from Deinococccus radiodurans has been expressed, purified and crystallized. Crystals were obtained by the microbatch-under-oil method at room temperature. The crystals diffracted to 2.36 A resolution at the Canadian Light Source. The space group was found to be P2(1)2(1)2(1), with unit-cell parameters a = 134.0, b = 176.6, c = 221.6 A. The initial structure solution was determined by molecular replacement using UGM from Mycobacterium tuberculosis (PDB code 1v0j) as a template model.

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Expression, purification and crystallization of two endonuclease III enzymes from Deinococcus radiodurans.

Project description:Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3?76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31?Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09?Å, ? = 89.34° and one molecule per asymmetric unit. The EndoIII-3?76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47?Å, ? = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3?76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

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High-resolution structure of the E.coli RecQ helicase catalytic core.

Project description:RecQ family helicases catalyze critical genome maintenance reactions in bacterial and eukaryotic cells, playing key roles in several DNA metabolic processes. Mutations in recQ genes are linked to genome instability and human disease. To define the physical basis of RecQ enzyme function, we have determined a 1.8 A resolution crystal structure of the catalytic core of Escherichia coli RecQ in its unbound form and a 2.5 A resolution structure of the core bound to the ATP analog ATPgammaS. The RecQ core comprises four conserved subdomains; two of these combine to form its helicase region, while the others form unexpected Zn(2+)-binding and winged-helix motifs. The structures reveal the molecular basis of missense mutations that cause Bloom's syndrome, a human RecQ-associated disease. Finally, based on findings from the structures, we propose a mechanism for RecQ activity that could explain its functional coordination with topoisomerase III.

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Crystallization and preliminary X-ray characterization of a catalytic and ATP-binding domain of a putative PhoR histidine kinase from the gamma-radioresistant bacterium Deinococcus radiodurans.

Project description:The gene product of histidine kinase DR2244 (putative phoR) encoded by Deinococcus radiodurans has been suggested to be involved in the PhoR-PhoB two-component regulatory system. This two-component signalling system is activated upon phosphate starvation in several bacteria, including D. radiodurans. Single crystals were obtained from a recombinant preparation of the catalytic/ATP-binding (CA) domain of D. radiodurans PhoR (79-224) overexpressed in Escherichia coli. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.9, b = 81.8, c = 204.6 A. The crystals contained six molecules in the asymmetric unit. Diffraction data were collected to 2.4 A resolution on beamline ID23-2 of the European Synchrotron Radiation Facility.

| S-EPMC2852330 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain.

Project description:DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P2(1), with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 A, alpha = gamma = 90, beta = 101.02 degrees , and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 A using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).

| S-EPMC2330201 | biostudies-literature

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