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Journal of Bacteriology, March 2002, p. 1649-1660, Vol. 184, No. 6
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.6.1649-1660.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Jong-Il Kim,1,{dagger} Ajay K. Sharma,2,{ddagger} Stephen N. Abbott,1 Elizabeth A. Wood,1 David W. Dwyer,1 Aaron Jambura,1 Kenneth W. Minton,2 Ross B. Inman,1,3 Michael J. Daly,2 and Michael M. Cox1*

Department of Biochemistry,1 Department of Molecular Virology, University of Wisconsin—Madison, Madison, Wisconsin 53706,3 Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 208142

Received 18 July 2001/ Accepted 2 November 2001

The RecA protein of Deinococcus radiodurans (RecADr) is essential for the extreme radiation resistance of this organism. The RecADr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecADr protein and the E. coli RecA (RecAEc) proteins are close functional homologues. RecADr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecAEc. The RecADr 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 RecADr 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 RecADr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecADr 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 RecADr protein binds much better to duplex DNA than the RecAEc 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.

* Corresponding author. Mailing address: Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Dr., Madison, WI 53706-1544. Phone: (608) 262-1181. Fax: (608) 265-2603. E-mail: cox{at}biochem.wisc.edu.

{dagger} Present address: Department of Applied Microbiology, Seoul Women's University, Nowon-Gu, Seoul, Korea.

{ddagger} Present address: Laboratory of Molecular Pharmacology, National Cancer Institute/NIH, Bethesda, MD 20892.

Journal of Bacteriology, March 2002, p. 1649-1660, Vol. 184, No. 6
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.6.1649-1660.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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