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Journal of Bacteriology, September 1998, p. 4879-4885, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Spore Photoproduct Lyase from Bacillus subtilis Spores Is a Novel Iron-Sulfur DNA Repair Enzyme Which Shares Features with Proteins such as Class III Anaerobic Ribonucleotide Reductases and Pyruvate-Formate Lyases

Roberto Rebeil,1 Yubo Sun,2,dagger Lilian Chooback,2,Dagger Mario Pedraza-Reyes,3 Cynthia Kinsland,4 Tadhg P. Begley,4 and Wayne L. Nicholson1,*

Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 857211; Department of Microbiology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas 761072; Facultad de Ciencias Químicas, Instituto de Investigación en Biología Experimental, Universidad de Guanajuato, Guanajuato, México3; and Chemistry Department, Cornell University, Ithaca, New York 148534

Received 8 June 1998/Accepted 20 July 1998

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned B. subtilis splB gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged SP lyase expressed from the amyE locus restored UV resistance to spores of a UV-sensitive mutant B. subtilis strain carrying a deletion-insertion mutation which removed the entire splAB operon at its natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant B. subtilis spores and from an Escherichia coli overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition of S-adenosylmethionine as a cofactor.

* Corresponding author. Mailing address: Department of Veterinary Science and Microbiology, Building 90, Room 102, University of Arizona, Tucson, AZ 85721. Phone: (520) 621-2157. Fax: (520) 621-6366. E-mail: WLN{at}u.arizona.edu.

dagger Present address: Department of Medicine, University of Miami, Coral Gables, FL 33124.

Dagger Present address: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019.

Journal of Bacteriology, September 1998, p. 4879-4885, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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