A soluble RecN homologue provides means for biochemical and genetic analysis of DNA double-strand break repair in Escherichia coli
| Autor: | Robert G. Lloyd, Stuart R. Wood, Jane I. Grove, Geoffrey S. Briggs, Neil J. Oldham |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
DNA Repair
DNA repair Molecular Sequence Data Bacillus subtilis medicine.disease_cause Biochemistry chemistry.chemical_compound Bacterial Proteins Escherichia coli medicine DNA Breaks Double-Stranded Amino Acid Sequence SOS response Molecular Biology Gene Adenosine Triphosphatases Aquifex aeolicus Sequence Homology Amino Acid biology DNA Restriction Enzymes Cell Biology biology.organism_classification Molecular biology Solubility chemistry Aquifex Protein Multimerization Sequence Alignment DNA |
| Zdroj: | DNA Repair. 8:1434-1443 |
| ISSN: | 1568-7864 |
| DOI: | 10.1016/j.dnarep.2009.09.015 |
| Popis: | RecN is a highly conserved, SMC-like protein in bacteria. It plays an important role in the repair of DNA double-strand breaks and is therefore a key factor in maintaining genome integrity. The insolubility of Escherichia coli RecN has limited efforts to unravel its function. We overcame this limitation by replacing the resident coding sequence with that of Haemophilus influenzae RecN. The heterologous construct expresses Haemophilus RecN from the SOS-inducible E. coli promoter. The hybrid gene is fully functional, promoting survival after I-SceI induced DNA breakage, gamma irradiation or exposure to mitomycin C as effectively as the native gene, indicating that the repair activity is conserved between these two species. H. influenzae RecN is quite soluble, even when expressed at high levels, and is readily purified. Its analysis by ionisation-mass spectrometry, gel filtration and glutaraldehyde crosslinking indicates that it is probably a dimer under physiological conditions, although a higher multimer cannot be excluded. The purified protein displays a weak ATPase activity that is essential for its DNA repair function in vivo. However, no DNA-binding activity was detected, which contrasts with RecN from Bacillus subtilis. RecN proteins from Aquifex aeolicus and Bacteriodes fragilis also proved soluble. Neither binds DNA, but the Aquifex RecN has weak ATPase activity. Our findings support studies indicating that RecN, and the SOS response in general, behave differently in E. coli and B. subtilis. The hybrid recN reported provides new opportunities to study the genetics and biochemistry of how RecN operates in E. coli. |
| Databáze: | OpenAIRE |
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