Browsing by Subject "spore photoproduct"

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    EPR Study of UV-irradiated Thymidine Microcrystals Supports Radical Intermediates in Spore Photoproduct Formation
    (ACS, 2016-09) Hayes, Ellen C.; Jian, Yajun; Stoll, Stefan; Li, Lei; Department of Physics, School of Science
    Spore photoproduct is a thymidine dimer formed when bacterial endospore DNA is exposed to ultraviolet (UV) radiation. The mechanism of formation of this thymidine dimer has been proposed to proceed through a radical-pair intermediate. The intermediate forms when a methyl-group hydrogen atom of one thymidine nucleobase is transferred to the C6 position of an adjacent thymidine nucleobase, forming two species, the TCH2 and TH radicals, respectively. Using a series of thymidine isotopologues and electron paramagnetic resonance (EPR) spectroscopy, we show that microcrystals of thymidine exposed to UV radiation produce these two radical species. We observe three sources that donate the additional hydrogen at the C6 position of the TH radical. One of the three sources is the methyl group of another thymidine molecule in a significant fraction of the TH species. This lends support to the radical-pair intermediate proposed in the formation of spore photoproduct.
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    Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme—The spore photoproduct lyase
    (Elsevier, 2017-04) Yang, Linlin; Jian, Yajun; Setlow, Peter; Li, Lei; Chemistry and Chemical Biology, School of Science
    DNA repair enzymes typically recognize their substrate lesions with high affinity to ensure efficient lesion repair. In UV irradiated endospores, a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, termed the spore photoproduct (SP), is the dominant DNA photolesion, which is rapidly repaired during spore outgrowth mainly by spore photoproduct lyase (SPL) using an unprecedented protein-harbored radical transfer process. Surprisingly, our in vitro studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01–0.2 min−1. The faster turnover numbers are reached under single turnover conditions, and SPL activity is low with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3–0.4 min−1, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from other specialized DNA repair enzymes. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins, helicases or an altered DNA conformation may cooperate with SPL to enable efficient SP repair in germinating spores. Therefore, both SP formation and SP repair are likely to be tightly controlled by the unique cellular environment in dormant and outgrowing spore-forming bacteria, and thus SP repair may be extremely slow in non-spore-forming organisms.
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    Using Organic Synthesis and Chemical Analysis to Understand the Photochemistry of Spore Photoproduct and Other Pyrimidine Dimers
    (Thieme, 2018) Li, Lei; Chemistry and Chemical Biology, School of Science
    Pyrimidine dimerization is the dominant DNA photoreaction occurring in vitro and in vivo. Three types of dimers, cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), and the spore photoproduct (SP), are formed from the direct dimerization process; it is of significance to understand the photochemistry and photobiology of these dimers. Traditionally, pyrimidine dimerization was studied by using the natural pyrimidine residues thymine and cytosine, which share similar chemical structures and similar reactivity, making it sometimes less straightforward for one to identify the key pyrimidine residue that needs to be excited to trigger the photoreaction. We thus adopted synthetic chemistry to selectively modify the pyrimidine residues or to introduce pyrimidine analogs to the selected positions before UV irradiation is applied. By monitoring the subsequent outcomes from the photoreaction, we were able to gain unique mechanistic insights into the photochemistry of SP as well as of CPDs and 6-4PPs. Moreover, our approaches have resulted in several useful “tools” that can facilitate the understanding of lesion photobiology. Our results summarized in this account illustrate what organic synthesis/chemical analysis may allow us to achieve in future DNA lesion biology studies.