RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans : Expression, Purification, and Characterization

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Abstract
The RecA protein of Deinococcus radiodurans (RecA Dr ) is essential for the extreme radiation resistance of this organism. The RecA Dr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA Dr protein and the E. coli RecA (RecA Ec ) proteins are close functional homologues. RecA Dr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA Ec . The RecA Dr protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA Dr protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA Dr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA Dr protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA Dr protein binds much better to duplex DNA than the RecA Ec protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus .