This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Slieman, T. A.
Right arrow Articles by Nicholson, W. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Slieman, T. A.
Right arrow Articles by Nicholson, W. L.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2000, p. 6412-6417, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Spore Photoproduct (SP) Lyase from Bacillus subtilis Specifically Binds to and Cleaves SP (5-Thyminyl-5,6-Dihydrothymine) but Not Cyclobutane Pyrimidine Dimers in UV-Irradiated DNA

Tony A. Slieman,dagger Roberto Rebeil, and Wayne L. Nicholson*

Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 85721

Received 17 April 2000/Accepted 22 August 2000

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Pydiamond Py) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Pydiamond Py-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the 32P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.

* Corresponding author. Mailing address: Department of Veterinary Science and Microbiology, 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 Biology, Morningside College, Sioux City, IA 51106.

Journal of Bacteriology, November 2000, p. 6412-6417, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Moeller, R., Setlow, P., Reitz, G., Nicholson, W. L. (2009). Roles of Small, Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation. Appl. Environ. Microbiol. 75: 5202-5208 [Abstract] [Full Text]  
  • Chandor-Proust, A., Berteau, O., Douki, T., Gasparutto, D., Ollagnier-de-Choudens, S., Fontecave, M., Atta, M. (2008). DNA Repair and Free Radicals, New Insights into the Mechanism of Spore Photoproduct Lyase Revealed by Single Amino Acid Substitution. J. Biol. Chem. 283: 36361-36368 [Abstract] [Full Text]  
  • Moeller, R., Setlow, P., Horneck, G., Berger, T., Reitz, G., Rettberg, P., Doherty, A. J., Okayasu, R., Nicholson, W. L. (2008). Roles of the Major, Small, Acid-Soluble Spore Proteins and Spore-Specific and Universal DNA Repair Mechanisms in Resistance of Bacillus subtilis Spores to Ionizing Radiation from X Rays and High-Energy Charged-Particle Bombardment. J. Bacteriol. 190: 1134-1140 [Abstract] [Full Text]  
  • Chandor, A., Berteau, O., Douki, T., Gasparutto, D., Sanakis, Y., Ollagnier-de-Choudens, S., Atta, M., Fontecave, M. (2006). Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis. J. Biol. Chem. 281: 26922-26931 [Abstract] [Full Text]  
  • Buis, J. M., Cheek, J., Kalliri, E., Broderick, J. B. (2006). Characterization of an Active Spore Photoproduct Lyase, a DNA Repair Enzyme in the Radical S-Adenosylmethionine Superfamily. J. Biol. Chem. 281: 25994-26003 [Abstract] [Full Text]  
  • Rebeil, R., Nicholson, W. L. (2001). The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase. Proc. Natl. Acad. Sci. USA 10.1073/pnas.161278998v1 [Abstract] [Full Text]  
  • Rebeil, R., Nicholson, W. L. (2001). The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase. Proc. Natl. Acad. Sci. USA 98: 9038-9043 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»