Characterization of the DNA damage-inducible helicase DinG from Escherichia coli
| Autor: | Filip Vanevski, Pavel P. Khil, Oleg N. Voloshin, R. Daniel Camerini-Otero |
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| Rok vydání: | 2003 |
| Předmět: |
Time Factors
DNA Repair Transcription Genetic DNA damage Cell Survival Ultraviolet Rays Amino Acid Motifs Molecular Sequence Data medicine.disease_cause Biochemistry Models Biological chemistry.chemical_compound Plasmid Adenosine Triphosphate Cations medicine Escherichia coli Amino Acid Sequence Molecular Biology biology Base Sequence Dose-Response Relationship Drug Escherichia coli Proteins Hydrolysis DNA replication Helicase Dose-Response Relationship Radiation Cell Biology DNA Hydrogen-Ion Concentration Molecular biology DNA helicase activity Kinetics Phenotype chemistry biology.protein Chromatography Gel Electrophoresis Polyacrylamide Gel Gene Deletion Genome Bacterial Nucleotide excision repair Plasmids |
| Zdroj: | The Journal of biological chemistry. 278(30) |
| ISSN: | 0021-9258 |
| Popis: | The dinG promoter was first isolated in a genetic screen scoring for damage-inducible loci in Escherichia coli (Lewis, L. K., Jenkins, M. E., and Mount, D. W. (1992) J. Bacteriol. 174, 3377–3385). Sequence analysis suggests that the dinG gene encodes a putative helicase related to a group of eukaryotic helicases that includes mammalian XPD (Koonin, E. V. (1993) Nucleic Acids Res. 21, 1497), an enzyme involved in transcription-coupled nucleotide excision repair and basal transcription. We have characterized the dinG gene product from E. coli using genetic and biochemical approaches. Deletion of dinG has no severe phenotype, indicating that it is non-essential for cell viability. Both dinG deletion and over-expression of the DinG protein from a multicopy plasmid result in a slight reduction of UV resistance. DinG, purified as a fusion protein from E. coli cells, behaves as a monomer in solution, as judged from gel filtration experiments. DinG is an ATP-hydrolyzing enzyme; single-stranded (ss) DNA stimulates the ATPase activity 15-fold. Kinetic data yield a Hill coefficient of 1, consistent with one ATP-hydrolyzing site per DinG molecule. DinG possesses a DNA helicase activity; it translocates along ssDNA in a 5′ → 3′ direction, as revealed in experiments with substrates containing non-natural 5′–5′ and 3′–3′ linkages. The ATP-dependent DNA helicase activity of DinG requires divalent cations (Mg2+, Ca2+, and Mn2+) but is not observed in the presence of Zn2+. The DinG helicase does not discriminate between ribonucleotide and deoxyribonucleotide triphosphates, and it unwinds duplex DNA with similar efficiency in the presence of ATP or dATP. We discuss the possible involvement of the DinG helicase in DNA replication and repair processes. |
| Databáze: | OpenAIRE |
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