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.Item DNA binding proteins of archaeal viruses(Montana State University - Bozeman, College of Letters & Science, 2008) Geary, Joella Suzanne; Chairperson, Graduate Committee: Mensur DlakicArchaea are single-celled organisms comprising the third domain of life. The Achaeal species Sulfolobus are infected by the Fuselloviridae virus family: SSV1, SSV2, SSV-RH, and SSV-K. The genomes of these viruses have been annotated and contain putative DNA-binding proteins. The purpose of this work is to identify DNA sequences bound by the SSV1 putative DNA-binding protein C43. C43 protein was cloned, expressed, purified, and assayed at various temperatures for interaction with three SSV1 DNA sequences. C43 binds the T5-promoter, T6-promoter, and C43-promoter sequentially and consistently. Additionally, C43 protein is functional at temperatures of 50°C and 65°C. Thus, C43 appears to be an important regulator of the Fuselloviridae SSV1 viral genome.Item X-ray crystallographic studies of the proteins from sulfolobus spindle-shaped viruses (SSVs)(Montana State University - Bozeman, College of Letters & Science, 2009) Menon, Smita Kesavankutty; Chairperson, Graduate Committee: C. Martin LawrenceViruses populate virtually every ecosystem on the planet. Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world. However, compared to eukaryotic and bacterial viruses, our knowledge of viruses infecting the archaea is limited. Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, structural studies can reveal distant evolutionary relationships and provide functional insights that are not apparent from the primary amino acid sequence alone. Several such structural studies have already contributed to our understanding of the Sulfolobus Spindle-shaped viruses (Fuselloviridae). Here we report the structure of two proteins, SSV1 F112 and SSVRH D212. Biochemical, proteomic and structural studies of F112 reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Continuing these efforts, a second structure was also determined where the overall fold and conservation of active site residues place D212 within the PD-(D/E)XK nuclease superfamily. Notably, the structure of F112 contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses and the evolutionary implications of such distribution are discussed. Here we review and describe our progress towards understanding these viruses at a molecular level.