Repair of UV-induced DNA damage is central to the prevention of a number of adverse conditions such as melanoma, but in most cases the mechanism of such DNA repair is not well understood at a molecular level. Under normal cellular conditions, the major DNA photoproducts of UV irradiation are cyclobutane pyrimidine dimers (TT, CT, and CC dimers) and 6,4-photoproducts. 5-Thyminyl-5,6-dihydrothymine is typically a minor product of UV irradiation. This typically minor UV photoproduct becomes the major UV photoproduct under certain conditions, however, including the conditions that exist in bacterial spores (thus the common name is spore photoproduct, SP). Remarkably, the formation of spore photoproduct is correlated with the unusually high resistance of bacterial spores to UV irradiation, and this resistance arises in part from the novel DNA repair enzyme that repairs SP. The overall goals of this proposal include investigating the mechanism by which the repair enzyme, SP lyase, recognizes and repairs SP. In addition, experiments designed to probe the mechanism by which SP is formed at the expense of cyclobutane pyrimidine dimers, are described.
The specific aims of this proposal are as follows: 1) To investigate the structure and environment of the iron-sulfur cluster in spore photoproduct lyase using appropriate spectroscopic methods; 2) To probe the mechanistic details of the DNA repair reaction using isotope labeling and substrate analogs; 3) To investigate the molecular basis of the interaction of SPL with UV-damaged DNA using gel-shift assays and DNA footprinting; 4) To investigate the interaction between the iron-sulfur cluster of SP lyase and its substrates S-adenosylmethionine and spore photoproduct using Mossbauer, electron-nuclear double resonance, and electron-spin echo envelope modulation; and 5) To investigate the structural basis of SP formation using biochemical and X-ray structural methods. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM067804-01
Application #
6599550
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
2003-05-01
Project End
2007-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
1
Fiscal Year
2003
Total Cost
$250,271
Indirect Cost
Name
Michigan State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Silver, Sunshine C; Gardenghi, David J; Naik, Sunil G et al. (2014) Combined Mössbauer spectroscopic, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of the radical SAM enzyme spore photoproduct lyase. J Biol Inorg Chem 19:465-83
Silver, Sunshine C; Chandra, Tilak; Zilinskas, Egidijus et al. (2010) Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase. J Biol Inorg Chem 15:943-55
Chandra, Tilak; Broderick, William E; Broderick, Joan B (2010) An efficient deprotection of N-trimethylsilylethoxymethyl (SEM) groups from dinucleosides and dinucleotides. Nucleosides Nucleotides Nucleic Acids 29:132-43
Chandra, Tilak; Silver, Sunshine C; Zilinskas, Egidijus et al. (2009) Spore photoproduct lyase catalyzes specific repair of the 5R but not the 5S spore photoproduct. J Am Chem Soc 131:2420-1
Duschene, Kaitlin S; Veneziano, Susan E; Silver, Sunshine C et al. (2009) Control of radical chemistry in the AdoMet radical enzymes. Curr Opin Chem Biol 13:74-83
Chandra, Tilak; Broderick, William E; Broderick, Joan B (2009) Chemoselective deprotection of triethylsilyl ethers. Nucleosides Nucleotides Nucleic Acids 28:1016-29
Buis, Jeffrey M; Cheek, Jennifer; Kalliri, Efthalia et al. (2006) Characterization of an active spore photoproduct lyase, a DNA repair enzyme in the radical S-adenosylmethionine superfamily. J Biol Chem 281:25994-6003