Crystallization and preliminary X-ray crystallographic and electron microscopic study of a bacterial DNA helicase (RSF1010 RepA)

Röleke, D.; Hoier, Helga; Bartsch, Cornelia; Umbach, Patrick; Scherzinger, Eberhard

doi:10.1107/s0907444996012565

Cited by 21 publications

(47 citation statements)

References 2 publications

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“…3B) shows that no other symmetry component presents a significant energy, apart from the dominant 6-fold harmonic. This fits well with the available crystallographic data that support a macromolecular architecture for RepA with 6-fold rotational symmetry (15).…”

Section: Fig 2 Subunit Composition Of the Purified Repa Proteinsupporting

confidence: 70%

“…At pH 5.6, the hexamers aggregate to form tubular structures. Crystallization experiments of RepA resulted in crystals diffracting to 3.5-Å resolution (15). These data demonstrated the presence of a 6-fold rotational axis within the molecule so that the six monomers are oriented identically to each other.…”

mentioning

confidence: 76%

“…The property of RepA to form stable hexamers in the absence of cofactors probably has facilitated the growth of diffracting crystals suitable for x-ray analysis (15) and subsequent structure refinement.…”

Section: Discussionmentioning

confidence: 99%

“…Micrographs of RepA helicase at pH 7.0 were prepared as described (15). They were digitalized in an Eikonix camera with a pixel size of 3.8 Å and subsequently processed using the Xmipp package (39).…”

Section: Image Reconstructionmentioning

confidence: 99%

“…However, the biological role of this monomeric protein derived from Bacillus stearothermophilus remains unclear. Another recent report described preliminary x-ray crystallographic data of the first hexameric replicative helicase crystallized, the RepA protein of plasmid RSF1010 (15), which is one of the smallest helicases known (29,896 Da/subunit). Structural analysis using electron microscopy revealed that, at neutral pH, RepA is a ring-shaped hexamer with a central channel formed by the globular subunits (15).…”

mentioning

confidence: 99%

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The RepA Protein of Plasmid RSF1010 Is a Replicative DNA Helicase

Scherzinger

Ziegelin

Bárcena

et al. 1997

Journal of Biological Chemistry

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The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication. RepA is one of the smallest known helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of RepA. Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from RepA crystals diffracting at 3.5-Å resolution (Rö leke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W. (1997) Acta Crystallogr. Sec. D 53, 213-216). The RepA helicase has 5 3 3 polarity. As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3-tail mimicking a replication fork. Optimal unwinding activity was achieved at the remarkably low pH of 5.5. In the presence of Mg 2؉ (Mn 2؉ ) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not. The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein.

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Section: Fig 2 Subunit Composition Of the Purified Repa Proteinsupporting

confidence: 70%

mentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Image Reconstructionmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The RepA Protein of Plasmid RSF1010 Is a Replicative DNA Helicase

Scherzinger

Ziegelin

Bárcena

et al. 1997

Journal of Biological Chemistry

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Rubromycins: Structurally Intriguing, Biologically Valuable, Synthetically Challenging Antitumour Antibiotics

et al. 2007

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Although known for more than 50 years the rubromycin family still constitutes a fascinating class of antitumour antibiotics. They are characterized by a challenging molecular architecture with the central spiroketal unit as the key feature and possess highly attractive biological properties. After a short treatment of the history of their isolation, structural elucidation and biosynthesis, their biological activities will briefly be summarized. This review strongly emphasizes the synthetic efforts aimed at these complex hexacyclic spiroketals. Reactions leading to simple spiroketal model compounds are described, followed by the synthetic approaches to the fully functionalized naphthalene and isocoumarin “wings”. The coupling of these units and their transformations into more advanced spiroketals demonstrate “the state of the art” in this research field. Only Danishefsky and co‐workers have so far completed the total synthesis of a fully functionalized rubromycin derivative; however, their product heliquinomycinone (103) is still only the aglycon of the natural product heliquinomycin (7), and it was prepared as the racemic compound. All these achievements and pitfalls reveal that increased engagement of synthetic organic chemists is required to develop new methods to make rubromycins and their analogues available by a modular approach and with reasonable efficacy. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

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