Theses and Dissertations at Montana State University (MSU)
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Item Binding and repair of DNA by spore photoproduct lyase(Montana State University - Bozeman, College of Letters & Science, 2010) Zilinskas, Egidijus; Chairperson, Graduate Committee: Joan B. BroderickBacterial spores are extremely resistant to chemical and physical stresses, including UV irradiation, which in spores results in the formation of 5-thyminyl-5,6-dihydrothymine (spore photoproduct, SP). While SP accumulates in UV-irradiated bacterial spores, it is rapidly repaired during germination. Spore photoproduct lyase (SPL) is the enzyme that catalyzes the specific repair of spore photoproduct to two thymines. It utilizes S-adenosylmethionine (SAM) and a [4Fe-4S] cluster to catalyze this reaction, and is a member of the radical SAM superfamily. Presented here is an investigation of SPL repair activity towards stereochemically-defined synthetic R- and Sspore photoproduct dinucleosides and dinucleotides (SP and SPTpT, respectively), utilizing SPL purified from Clostridium acetobutylicum. The results of HPLC and Mass Spectrometry analysis of in vitro enzymatic assays demonstrate that SPL specifically repairs the 5R-, but not the 5S- isomer. The repair rates were determined to be ~0.4 nmol/min/mg of SPL for the 5R-SP dinucleoside and ~7.1 nmol/min/mg of SPL for the 5R-SPTpT dinucleotide. Since SPL binding to DNA is a key step in UV damage repair, SPL binding to undamaged DNA, as well as the 5R- and the 5S-isomers of SP and SPTpT, was also investigated. The binding to different substrates was investigated by carrying out electrophoretic mobility shift assays (EMSA) and time-resolved fluorescence decay experiments. SPL from both Bacillus subtilis and Clostridium acetobutylicum cooperatively binds the undamaged DNA with relatively high affinity (Kd = 4.7 x 10 -9 M for B.s. SPL and Kd = 1.7 x 10 -7 M for C.a. SPL). The presence of small, acid-soluble proteins (SASP), SAM or the [4Fe-4S] cluster of SPL have little effect on SPL binding to undamaged DNA. Interestingly, SPL is able to bind both the 5R- and the 5S- diastereomers of the synthetic dinucleoside/dinucleotide spore photoproduct, although only the 5R-isomer is repaired. SP lyase binding is stronger to the SPTpT dinucleotide than to the SP dinucleoside, likely due to the dinucleotide more closely resembling the natural substrate in double helical DNA. Also, SPL exhibits higher affinity towards SP and SPTpT than the repair products, thymidine or thymidylyl (3'-5') thymidine (TpT), respectively.