The RadB protein from Pyrococcus does not complement E. coli recA mutations in vivo
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O R I GI N A L P A P E R
W. Inwood á S. Kane á J. DiRuggiero á F. Robb A. J. Clark
The RadB protein from Pyrococcus does not complement E. coli recA mutations in vivo Received: 18 December 2000 / Accepted: 23 January 2001 / Published online: 10 April 2001 Ó Springer-Verlag 2001
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 arti®cial 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. Keywords radB gene á Pyrococcus furiosus á recA gene á Recombination á Repair of UV damage
Introduction The RecA recombinase or one of its homologs is present in all free-living organisms, where it carries out the synapsis step of homologous recombination and recombiCommunicated by R. Devoret W. Inwood á S. Kane á A. J. Clark (&) Department of Molecular and Cell Biology, Division of Life Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA E-mail: [email protected] Tel.: +1-520-626-5437 Fax: +1-520-621-3709 J. DiRuggiero á F. Robb Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA A. J. Clark Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA
national DNA repair (Roca and Cox 1997; Bianco et al. 1998; Eisen and Hanawalt 1999). Among the Archaea, the putative recombinase homolog is called RadA (Sandler et al. 1996). The in vitro activity of RadA protein is very similar to that of RecA and also that of the homolog from Saccharomyces cerevisiae called Rad51 (Seitz et al. 1998; Seitz and Kowalczykowski 2000; Komori et al. 2000). A second homolog, named RadB, has also been identi®ed in Archaea (Rashid et al. 1996; DiRuggiero et al. 1999). An in vivo experiment yielded results that were interpreted as indicating that RadB protein could functionally replace RecA in a recA mutant strain of E. coli (Rashid et al. 1996). However, the in vitro activity reported for puri®ed RadB protein indicated that it possessed nuclease activities (Rashid et al. 1997, 1999), which are not associated with RecA and Rad51 proteins (Bianco et al. 1998; Zaitseva et al. 1999). We set out to con®rm the results with RadB because we thought that RadB might represent a primitive recombinase, which could carry out exonucleolytic presynaptic and endonucleolytic postsynaptic steps, as well as the synaptic step that RecA homologs are known to catalyze. We drew support for this hypothesis from the alignment of RadB protein with the common portio
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