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Journal of Bacteriology, October 2008, p. 6475-6482, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.01165-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Stable, Functional Core of DdrA from Deinococcus radiodurans R1 Does Not Restore Radioresistance In Vivo{triangledown}

Dennis R. Harris, Khanh V. Ngo, and Michael M. Cox*

Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin

Received 23 July 2007/ Accepted 18 July 2008

DdrA protein binds to and protects 3' DNA ends and is essential for preserving the genome integrity of Deinococcus radiodurans following treatment by gamma radiation in an environment lacking nutrients. Limited proteolysis was used to identify a stable and functional protein core, designated DdrA157, consisting of the first 157 residues of the protein. In vitro, the biochemical differences between wild-type and mutant proteins were modest. DdrA exhibits a strong bias in binding DNA with 3' extensions but not with 5' extensions. The mutant DdrA157 exhibited a greater affinity for 5' DNA ends but still bound to 3' ends more readily. However, when we replaced the wild-type ddrA gene with the mutant gene for ddrA157, the resulting D. radiodurans strain became almost as sensitive to gamma radiation as the ddrA knockout strain. These results suggest that while the stable protein core DdrA157 is functional for DNA binding and protection assays in vitro, the carboxyl terminus is required for important functions in vivo. The C terminus may therefore be required for protein or DNA interactions or possibly as a regulatory region for DNA binding or activities not yet identified.

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

{triangledown} Published ahead of print on 1 August 2008.

Journal of Bacteriology, October 2008, p. 6475-6482, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.01165-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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