▼ The process of DNA replication can be hindered by DNA damage, and disruptions in replication ultimately lead to cell death if not corrected. Bacteria solve this problem with a mechanism called “DNA replication restart,” which is catalyzed by primosome proteins. However, not all bacteria encode the full complement of primosome proteins found in the well-studied bacterium, E. coli. This suggests that differences might exist in DNA replication restart pathways among diverse bacteria. N. gonorrhoeae, for example, lacks a DnaT homolog. DnaT is thought to affect interactions between primosome protein PriB and single-stranded DNA. Since N. gonorrhoeae PriB has a weak interaction with single-stranded DNA, as opposed to the strong interaction between PriB and single-stranded DNA seen in E. coli (which encodes a DnaT homolog), we hypothesized that the presence of a DnaT homolog could be used to predict the affinity with which a bacterial PriB binds single-stranded DNA. Binary interactions between PriB and single-stranded DNA of two bacteria, K. pneumoniae (which encodes a DnaT homolog) and Y. enterocolitica (which lacks a DnaT homolog) were analyzed. Both K. pneumoniae and Y. enterocolitica have a high affinity PriB:ssDNA interaction with dissociation constants of 62 ± 14 nM and 84 ± 8 nM, respectively. Thus, the presence of DnaT cannot be used to predict affinities of binary interactions between PriB and single-stranded DNA. However, the experimentally measured binding constants combined with amino acid sequence alignments of the PriB homologs have led to the definition of parameters for high affinity binary interactions between PriB and single-stranded DNA. A recent report describes the identification of a novel PriB protein in K. pneumoniae that is significantly shorter than most sequenced PriB homologs (H.C. Hsieh, and C.Y. Huang, Identification of a novel protein, PriB, in Klebsiella pneumoniae. Biochem Biophys Res Commun 404 (2011) 546-51). The K. pneumoniae PriB protein is proposed to comprise 55 amino acid residues, in contrast to E. coli PriB which comprises 104 amino acid residues and has a length that is typical of most sequenced PriB homologs. Our investigations suggest that the priB gene of K. pneumoniae encodes a 104-amino acid PriB protein, akin to its E. coli counterpart. We have cloned the K. pneumoniae priB gene and purified the 104-amino acid K. pneumoniae PriB protein. Gel filtration experiments reveal that the K. pneumoniae PriB protein is a dimer, and equilibrium DNA binding experiments demonstrate that K. pneumoniae PriB's single-stranded DNA-binding activity is similar to that of E. coli PriB. These results indicate that the PriB homolog of K. pneumoniae is similar in structure and in function to that of E. coli. Advisors/Committee Members: Lopper, Matthew E.

▼ DNA replication restart pathways enable bacterial cells to reinitiate DNA replication when replication has been disrupted due to encounters with DNA damage, thereby allowing complete and faithful duplication of the cell's genetic information. Neisseria gonorrhoeae is a bacterium that is highly adapted to survive oxidative damage to its DNA incurred by attack from immune cells in infected individuals, suggesting that DNA replication restart pathways might play a critical role in N. gonorrhoeae pathogenicity. The bacterial helicase, PriA, is a key primosome protein that plays essential roles in DNA replication restart pathways. However, little is known of the mechanism by which PriA performs these roles in N. gonorrhoeae. I performed equilibrium DNA binding assays and DNA unwinding assays to provide insight into the mechanisms by which PriA functions in DNA replication restart pathways. I report that DNA binding by PriA is strongly dependent on the structure of the DNA. DNA substrates that resemble a DNA replication fork with a three-way branch are bound with higher affinity than partial duplex structures or single-stranded DNA. PriA-catalyzed DNA unwinding is also DNA structure-specific, and PriA-catalyzed unwinding decreases upon increasing the length of the duplex DNA, indicating that PriA is a low-processivity helicase. Another primosome protein, PriB, strongly stimulates the helicase activity of PriA, and this activity might facilitate reloading of the replication machinery by PriA at repaired replication forks. Stimulation of PriA by PriB appears to occur through a mechanism that is distinct from that used by the well-studied E. coli primosome proteins. Advisors/Committee Members: Lopper, Matthew.

▼ Neisseria gonorrhoeae is the causative organism of gonorrheal infection. This pathogen shows remarkable resistance to the oxidative damaging agents released by the neutrophils. The Bacterial genome is one of the important targets of these agents and studies indicate that DNA replication restart pathways help in bypassing the hazardous effects of these oxidative agents. The DNA replication restart pathway helps to reload and restart the stalled or derailed replication machinery onto the DNA which is blocked by obstructions such as single stranded nicks, double stranded breaks, or oxidized bases. In N. gonorrhoeae, the primosome proteins PriA and PriB carry out the DNA replication restart process by forming a nucleoprotein complex with the DNA. PriA, a helicase protein, binds and partially unwinds the DNA at the fork where the replication machinery will be reloaded. PriB serves to stimulate PriAs DNA binding and unwinding activity. PriA, PriB and the DNA form a ternary nucleo-protein complex in this process. In, E. coli the PriB has a high affinity interaction with ssDNA and a low affinity interaction with PriA, while N. gonorrhoeae PriB has a high affinity interaction with PriA and a low affinity interaction with ssDNA. The first part of my research contributed to understand the features of this affinity reversal. I performed pull down experiments, DNA unwinding assays and ATP hydrolysis experiments using single point alanine mutants of PriB. From my work, I have provided evidence that in N. gonorrhoeae, PriBs ssDNA binding activity is not required for PriB stimulation of PriA helicase, unlike what is seen in E. coli. Evidence that PriA provides resistance against oxidative damaging agents bolsters the importance of DNA replication restart pathway for the survival of this disease causing bacteria. Developing antibiotics that target this pathway could be a vital event in the field of drug discovery research. In this process, we have developed an enzyme-based assay to use in high-throughput screening to identify inhibitors of the DNA replication restart pathway in N. gonorrhoeae. Initial screening of several thousand compounds from small molecule chemical libraries has produced several lead compounds. I worked with one of the lead compounds, CGS-15493, and here I report the mechanistic features of its inhibition of the DNA replication restart pathway. Advisors/Committee Members: Lopper, Matthew E.

▼ Coordination of two [Ru(bipy)2Cl]+ moieties (where bipy = 2,2′-bipyridine) to the pyridyl nitrogens in the 5,10-positions of meso-5,10,15-(4-pyridyl)-20-(pentafluorophenyl)porphyrin gives the diruthenium porphyrin complex II. Insertion of copper(II) into the porphyrin center allows for the third pyridyl nitrogen to be coordinated to Pt(dmso)Cl2. Electronic transitions associated with the ruthenium porphyrin include an intense Soret band and four less intense Q-bands in the visible region of the spectrum. An intense π-π* transition in the UV region associated with the bipyridyl groups and a metal to ligand charge transfer (MLCT) band appearing as a shoulder to the Soret band are also observed. A slight blue shift of the Soret band and collapse of the Q-bands into one band is observed upon insertion of Cu(II) into the porphyrin center. No change in the electronic spectrum is observed upon coordination of the Pt(dmso)Cl2 moiety. Electrochemical properties associated with the complexes include a redox couple in the cathodic region attributed to the porphyrin and a redox couple in the anodic region due to the RuIII/II couple. DNA titrations of the complex III and IV indicate that both complexes interact strongly with DNA potentially through a partial intercalation mechanism. Gel electrophoresis studies indicate that complex IV has a greater effect on DNA migration through the gel than the well-known DNA binding agent cis-platin. Irradiation of aqueous solutions of complex III and supercoiled DNA at a 5:1 base pair to complex ratio (in the absence of oxygen) with visible light above 400 nm shows a nicking of the DNA. Repeat experiments in the presence of oxygen show that the complex III photocleaves the DNA, giving the linear form, as evidenced by gel electrophoresis. The cell studies show that the diruthenium porphyrin, complex II, is very toxic to the melanoma cells but appears to be overly toxic to normal cells when irradiated to be a feasible photosensitizer for PDT. However, only a little or no effect on the normal fibroblast or melanoma cells was observed after an irradiation time of 60 minutes for complexes III or IV. Advisors/Committee Members: Swavey, Shawn.

▼ β-carotene is comprised of isoprene units, and hence contain methyl-substituted polyene chromophores, which contains 11 highly unsaturated conjugated double bonds, nine of which are in a planar polyene chain and the other two of which are located in terminal β-ionylidene rings. The first objective is to determine the kinetics of β-carotene (βC) thermal and photo-degradation rate and evaluate the correlation among content, bleaching time and temperature and solvent effects. The second objective is to figure out the chemical structure of unknown thermal and photo-degradation extents and products structures by instruments of UV-Vis spectrometry, NMR and GC-MS. Solid βC was heated in sealed pyrex tubes to ensure isothermal heating and no oxygen impacts in open tubes with vents to detect oxygen impacts. Analysis of degradation kinetic data suggested a first-order reaction for the thermal-degradation of βC. The thermal-degradation of β-Carotene was studied at oven temperatures of 100, 150 , 200, 250, 300 , 350 °C for for 30min, 60min, 90min, 120min, 4h, 8h, 24h, 72h separately. The kinetics parameters, rate constant k, activation energy Ea =11.5763kJ/mol, and pre-exponential factor A (1.4041h-1) have been calculated based on the linear regression of concentration vs temperature (100-350°C). β-carotene is heat sensitive as expected and the degradation fate displayed a relative heat instability and temperature dependence. The higher the treatment temperature, the longer time exposure period, the faster the degradation rate. The degradation products, retinol, β- ionone etc are confirmed by GC-MS, most of other products are believed to be apocarotenals and epoxy compounds. The kinetics from photochemical experiments using UV irradiation are compared with similar experiments conducted with two-photon laser irradiation. The kinetics of photochemical reactions of β-carotene was studied in hexane, carbon tetrachloride and percentages of carbon tetrachloride in hexane below 5%. At low percentages of carbon tetrachloride, the reaction is first order in both carbon tetrachloride and β-carotene. The activation energy of the reactions at temperatures around 25°C was found to be positive but very small. The photochemical results are also compared with thermal-degradation experiments conducted at temperatures between 250 and 350°C. In addition to the kinetic results, some products of the reaction have been identified by nuclear magnetic resonance spectroscopy. It is interesting to note that under thermal conditions, retinal and retinol with vinyl hydrogens are identified as products, while under UV irradiation, there are no identifiable products that contain double bonds. Advisors/Committee Members: Johnson, David W.