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The RadB protein from Pyrococcus does not complement E. coli recA mutations in vivo
W Inwood1, S Kane, J DiRuggiero
1Department of Molecular and Cell Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Abstract:
A previous publication claimed that the radB gene called Pk-REC from Pyrococcus furiosus complemented an E. coli recA mutation. We found that a sequencing error had led to the test of a mutant form of Pk-REC. The wild-type radB gene from P. furiosus cloned in a similar expression vector to the mutant Pk-REC also appeared to complement an E. coli recA mutation. However, the cloned P. furiosus gdh (glutamate dehydrogenase) gene showed the same activity. We therefore concluded that overexpression of any protein can produce an artificial growth inhibition or stationary phase in recA mutant cells, which allows cells to recover from UV damage due to the action of repair systems that do not require RecA-like activity.
Insights
A previous study incorrectly claimed Pyrococcus furiosus Pk-REC complements E. coli recA mutations. Our findings show this was due to a sequencing error and artificial cell recovery from UV damage caused by protein overexpression.
Area of Science:
- Microbiology and Molecular Biology
- DNA Repair Mechanisms
- Extremophile Genetics
Background:
- Previous research suggested the Pyrococcus furiosus radB gene (Pk-REC) could complement Escherichia coli recA mutations.
- The recA gene is crucial for DNA repair in E. coli, particularly in response to UV radiation.
- Understanding complementation of recA mutations aids in deciphering DNA repair pathways across species.
Purpose of the Study:
- To re-evaluate the complementation of E. coli recA mutations by the P. furiosus radB (Pk-REC) gene.
- To investigate the underlying reasons for the previously reported complementation.
- To clarify the role of Pk-REC and potential artifacts in DNA repair studies.
Main Methods:
- Cloning and expression of wild-type and mutant Pk-REC genes from Pyrococcus furiosus in E. coli.
- Complementation assays using E. coli recA mutant strains.
- Testing the effect of expressing another P. furiosus gene (gdh) under similar conditions.
- UV irradiation survival assays for recA mutant cells.
Main Results:
- A sequencing error was identified, meaning the previously tested Pk-REC was a mutant form.
- Both the mutant and wild-type Pk-REC genes appeared to complement the E. coli recA mutation.
- Crucially, the cloned P. furiosus glutamate dehydrogenase (gdh) gene exhibited similar complementation activity.
- Overexpression of any protein, including Pk-REC and P. furiosus gdh, induced artificial growth recovery in recA mutant cells after UV damage.
Conclusions:
- The observed complementation of E. coli recA mutations by Pk-REC was an artifact, not true functional complementation.
- Protein overexpression in recA mutant cells can create a pseudo-recovery effect, masking the absence of RecA-like activity.
- This artifact allows for recovery from UV damage via RecA-independent repair pathways.
