Chemistry & Biochemistry Theses and Dissertations
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Item The role of sequence in the structure of self-assembling 3D DNA crystals(2015) Saoji, Maithili; Paukstelis, Dr. Paul J; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA is a widely used biopolymer for the construction of nanoscale objects due to its programmability and structural predictability. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. We previously determined the X-ray crystal structure of a DNA 13-mer that forms a continuously hydrogen bonded three-dimensional lattice through Watson-Crick and non-canonical base pairs. In the current work I examined how the sequence of the Watson-Crick duplex region influenced crystallization of this 13-mer. I screened all possible self-complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson-Crick base pair influenced the crystallization propensity and the speed of crystal self-assembly. I determined X-ray crystal structures for 13 of these oligonucleotides and found sequence-specific structural changes suggesting that this base pair may serve as a structural anchor during crystal assembly. I explored the crystal self-assembly and nucleation process and demonstrated that crystals grown from mixtures of two different oligonucleotide sequences contained both the oligonucleotides. These results suggested that crystal self-assembly is nucleated by the formation of Watson-Crick duplexes. Finally, I also examined how a single nucleotide addition to the DNA 13-mer leads to a significantly different overall structure under identical crystallization conditions. The 14-mer crystal structures described here showed that all of the predicted Watson-Crick base pairs were present, but the major difference as compared to the parent 13-mer structure was a significant rearrangement of non-canonical base pairs. This included the formation of a sheared A-G base pair, a junction of strands formed from base triple interactions, and tertiary interactions that generated structural features similar to tandem sheared G-A base pairs. The adoption of this alternate non-canonical structure was dependent in part on the sequence of the Watson-Crick duplex region. These results provided important new insights into the sequence/structure relationship of short DNA oligonucleotides and demonstrated a unique interplay between Watson-Crick and non-canonical base pairs that are responsible for crystallization fate.Item BIOLOGICAL CHARACTERIZATION OF TWO PUTATIVE DNA METABOLISM ENZYMES IN DEINOCOCCUS RADIODURANS(2014) Mueller, Charles; Julin, Douglas; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)HerA proteins are members of the FtsK-HerA superfamily of P-loop ATPases. FtsK is a bacterial protein that translocates double-stranded DNA during cell division. In archaea, herA is an essential gene that encodes an enzyme believed to be important for recombinational DNA repair. It is typically found in an operon with a gene that codes for a nuclease, nurA. Homologs of herA and nurA are found in a few bacterial genomes. In most cases, these bacteria lack an ftsK homolog. The functions of NurA and HerA in bacteria are not known. We chose to investigate the roles of NurA and HerA in Deinococcus radiodurans, typically studied for its extreme resistance to double-strand DNA breaks. The D. radiodurans genome has homologs of nurA and herA in an operon, and it also has an ftsK gene. We made strains with deletions of either herA or nurA and characterized their sensitivity to DNA damaging agents and basic growth properties. The results indicate that neither gene is essential in D. radiodurans, and deletions of the genes do not cause significant sensitivity to DNA damaging agents. The herA deletion strain displayed a distinct phenotype consisting of slower growth and larger cell types. The herA phenotype in D. radiodurans is similar to that of mutation of ftsK homologs in Escherichia coli and Bacillus subtilis. The results suggest that HerA has an FtsK-like function in cell division, rather than acting in DNA repair, in D. radiodurans.Item Jamming effects in glasses and biopolymers(2014) Kang, Hongsuk; Thirumalai, Devarajan; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this dissertation, jamming effects in highly packed systems are studied in two specific materials: glasses and biopolymers in cellular environments. Suspensions consisting of highly charged colloids, which are well-known glass-forming systems, are investigated using molecular dynamics simulations in order to test Random First Order Transition (RFOT) theory. I found that there is a critical volume fraction at which ergodic-to-nonergodic transitions for three dynamic observables take place in accordance with RFOT. Based on numerical observations, it is also proposed that the dynamic heterogeneity can be attributed to the violation of law of large numbers. In addition, the bond orientational order of colloidal suspensions and soft-spheres is discussed in the context of liquid-glass transitions. The response of biopolymers to a crowded environment is another interesting issue because 20-40% volume of a cell is occupied by various cellular components such as ribosomes and proteins in vivo. In this work, using low-friction langevin dynamics simulations with explicit crowding particles, I examined the conformational change of biopolymers in the presence of crowders of various sizes and shapes. The simulation results reveal that cylindrical crowders induce much greater compaction of the polymers than spherical ones at low volume fractions and the stronger crowding effects disappear at higher volume fractions due to local nematic ordering of cylindrical particles. The reduction in the size of polymer is even more dramatic in a mixture of spherical and cylindrical shapes because of cooperative crowding effects explained by the phase separation of spheres and rodlike particles. Finally, the crowding effects of cellular components on bacterial chromosomes are estimated using a mixture of spherical crowders with the composition found in bacterial cytoplasms.Item DESIGN AND DEVELOPMENT OF THREE-DIMENSIONAL DNA CRYSTALS UTILIZING CGAA PARALLEL BASE PAIRED MOTIFS(2013) Muser, Stephanie Elizabeth; Paukstelis, Paul; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Three-dimensional (3D) DNA crystals hold great potential for various applications such as the development of molecular scaffolds for use in protein structure determination by x-ray crystallography. The programmability and predictability of DNA make it a powerful tool for self-assembly but it is hindered by the linearity of the duplex structure. Predictable noncanonical base pairs and motifs have the potential to connect linear double-helical DNA segments into complex 3D structures. The sequence d(GCGAAAGCT) has been observed to form 3D crystals containing both noncanonical parallel pairs and canonical Watson-Crick pairs. This provided a template structure that we used in expanding the design and development of 3D DNA crystals along with exploring the use of predictable noncanonical motifs. The structures we determined contained all but one or two of the designed secondary structure interactions, depending on pH.Item Trapping Labile Adducts Formed Between an ortho-Quinone Methide and DNA(2012) McCrane, Michael Patrick; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exogenously generated electrophiles are capable of alkylating DNA. If not repaired, the resulting DNA adducts can lead to mutations and either cancer or cell death. Electrophilic ortho-quinone methides (o-QM) are reactive intermediates that alkylate DNA and are generated during xenobiotic metabolism of a variety of compounds including environmental toxins and therapeutic agents. Identifying the full alkylation profile of o-QM towards DNA would allow for the genotoxicity of o-QM precursors to be better understood. From model studies based on nucleosides, o-QMs react most readily, but reversibly with the strong nucleophiles 2'-deoxycytidine (dC) N3, 2'-deoxyguanosine (dG) N7, and 2'-deoxyadenosine (dA) N1 and less efficiently, but irreversibly with the weak nucleophiles dG N1, dG N2, and dA N6. The reverse reactions complicate analysis of their products in DNA, which requires enzymatic digestion and chromatographic separation. Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) can transform the reversible o-QM-DNA adducts into irreversible derivatives capable of surviving such analysis. To facilitate this analysis, a series of oxidized o-QM-dN adducts were synthesized as analytical standards. Initial oxidative trapping studies with an unsubstituted o-QM and dC demonstrated the necessity of an alkyl substituent para to the phenolic oxygen to block over-oxidation. A novel o-QM included a methyl group para to the phenolic oxygen that successfully blocked the over-oxidation allowing for generation of a stable MeQM-dC N3 oxidized product. Further oxidative trapping studies with MeQM and dG resulted in the formation of three stable MeQM-dG oxidized products (guanine N7, dG N1, and dG N2). Initial studies with duplex DNA optimized the enzymatic digestion and confirmed that the assay conditions were compatible with oxidative trapping. The low yielding MeQM alkylation of duplex DNA needs to be scaled up prior to the oxidative trapping studies. Alternative studies quantified the release of MeQM from DNA with the use of 2-mercaptoethanol as a nucleophilic trap. These studies revealed single stranded DNA as a superior carrier of MeQM than duplex DNA and, therefore, a better target DNA for the oxidative trapping studies due to increased yield of MeQM adducts. With the increased MeQM-DNA yield, the intrinsic selectivity and reactivity of MeQM towards DNA can be determined.Item MANIPULATION OF DNA TOPOLOGY USING AN ARTIFICIAL DNA-LOOPING PROTEIN(2012) Gowetski, Daniel; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA loop formation, mediated by protein binding, plays a broad range of roles in cellular function from gene regulation to genome compaction. While DNA flexibility has been well investigated, there has been controversy in assessing the flexibility of very small loops. We have engineered a pair of artificial coiled-coil DNA looping proteins (LZD73 and LZD87), with minimal inherent flexibility, to better understand the nature of DNA behavior in loops of less than 460 bp. Ring closure experiments (DNA cyclization) were used to observe induced topological changes in DNA upon binding to and looping around the engineered proteins. The length of DNA required to form a loop in our artificially rigid system was found to be substantially longer than loops formed with natural proteins in vivo. This suggests the inherent flexibility of natural looping proteins plays a substantial role in stabilizing small loop formation. Additionally, by incrementally varying the binding site separation between 435 bp and 458 bp, it was observed that the LZD proteins could predictably manipulate the DNA topology. At the lengths evaluated, the distribution of topological products correlates to the helical repeat of the double helix (10.5 bp). The dependence on binding site periodicity is an unequivocal demonstration of DNA looping and represents the first application of a rigid artificial protein in this capacity. By constructing these DNA looping proteins, we have created a platform for addressing DNA flexibility in regards to DNA looping. Future applications for this technology include a vigorous study of the lower limits of DNA length during loop formation and the use of these proteins in assembling protein:DNA nanostructures.Item DESIGN AND SYNTHESIS OF AN ELECTRON RICH QUINONE METHIDE PRECURSOR FOR SEQUENCE -DIRECTED ALKYLATION OF DNA(2012) Huang, Chengyun; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Quinone methides (QM) can be delivered to alklyate specific sites with a single strand DNA through target promoted alkylation. Previous experimental results indicated that alkylation by DNA-QM self-adduct was too slow for application in a biological system. A new quinone methide precursor (QMP) with enhanced reactivity is necessary to accelerate the reaction. Previous study showed that an electron donating group present in the QMP would facilitate the generation of QM from the precursor and its regeneration from the reversible alkylation adducts. Therefore, new QMPs with increased electron density were designed. An electron rich QMP2 was successfully synthesized through a benzylaldehyde derivative. As predicted, DNA-QM self-adduct formation was much faster using QMP2 than using the conventional precursor QMP1 without an electron donating group. Only 20 min was needed for QM2 to complete the conversion from DNA-QMP conjugate while QM1 needed 24 hrs to finish the same conversion. The DNA-QM2 self-adduct also exhibited faster reaction for alkylation of the target single strand. A two-day incubation was necessary to achieve its maximal yield of 20% compared to 6 days required to achieve the maximal yield of 16% for DNA-QM1. In order to target duplex DNA, QMP was coupled to triplex forming oligonucleotides (TFO) to deliver the QM to the major groove of DNA through triple helix formation. Alkylation products were observed with the DNA-QMP1 conjugate but not the DNA-QM1 self-adducts. An adjacent guanine in the sequence can increase alkylation yield from around 10% to up to 20%. QMP2 was also coupled to the TFO to generate the self-adduct DNA-QM2. Maximal duplex alkylation yield (15%) using DNA-QM2 self-adduct was achieved in 3 days if the triplex samples were incubated at room temperature. The alkylation yield increased to 20% with the DNA-QM2 self-adductwhen samples were incubated at 37 °C. The DNA-QMP2 conjugate could even be activated at 37 oC without fluoride and resulted in an alkylation yield of up to 25%. The enhanced reactivity of the electron rich QMP2 improved the duplex alkylation effectiveness and prepared it for future in vivo application.Item USING SINGLE MOLECULE TECHNIQUES TO DETERMINE THE MECHANISM OF DNA TOPOLOGY SIMPLIFICATION BY TYPE IIA TOPOISOMERASES(2011) Hardin, Ashley Harris; Thirumalai, Devarajan; Neuman, Keir C; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Type IIA topoisomerases are essential, universally conserved proteins that modify DNA topology by passing one segment of duplex DNA (the transfer, or T-segment) through a transient double strand break in a second segment of DNA (the gate, or G-segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot, and relax supercoiling in DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying non-equilibrium topology simplification remains speculative, though several plausible models have been proposed. This thesis tests two of these, the bend angle and kinetic proofreading models, using single-molecule techniques. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G-segment DNA by a type IIA topoisomerase. To test this model, we used atomic force microscopy and single molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that all proteins bent DNA, but the imposed bends are similar and cannot account for the differences among the enzymes. These data do not support the bend angle model and suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Based on the assumption that the rates of collision between DNA segments is higher in knotted, linked, and supercoiled DNA than in topologically free or relaxed DNA, the kinetic proofreading model proposes that two successive binding events between a G-segment bound topoisomerase and a putative T-segment are required to initiate strand passage. As a result of the two step process, the overall rate of strand passage should scale with the square of the collision probability of two DNA segments. To test this model, we used magnetic tweezers to manipulate a paramagnetic bead tethered to the surface by two DNA molecules. By rotating the bead, we varied the proximity, and thus collision rate, of the two molecules to determine the relationship between collision probability and rate of strand passage. Our data indicate that the strand passage rate scales linearly with the collision probability, which is inconsistent with the kinetic proofreading model.Item Fundamentals of Excess Electron Transport in Biologically Relevant Systems(2008-01-25) Finch, Amethist Shajary; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Thymine dimers are premutagenic lesions that form via ultraviolet irradiation of DNA. While the distribution of thymine dimers is non-random, it is also not yet predictable. Thymine dimer accumulation is likely to be controlled by both its formation and reversion. Formation of thymine dimers occurs via direct excitation of thymine residues, while the reversion is governed by both direct and indirect photochemical processes. The Rokita lab previously determined that charge transport from surrounding DNA sequences affect the accumulation of UV-induced thymine dimers in DNA. This dissertation focuses on the variable efficiency of both direct and indirect pathways affecting thymine dimer distribution. Thymine dimer accumulation is dependent upon sequence and conformation. The conformational dependence was used to study the kinetic versus thermodynamic control of thymine dimer accumulation in natural systems. In this system, no difference was observed in the accumulation of thymine dimer in free vs. constrained DNA. Therefore, the anticipated studies on the reversibility of thymine dimer formation still await a suitable system that does respond to DNA conformation. A model system was also used to concurrently investigate the effect of local sequence on thymine dimer accumulation. Excess electron transport in DNA is also important in modulating the overall accumulation of thymine dimers. The parameters affecting excess electron transport were investigated with a model system based on electron transfer from an aromatic amine to bromouridine. Electrons injected into duplex DNA by the aromatic amine migrate through the stacked nucleotides to the bromouridine acceptor covalently attached to the DNA. Modifications of the intervening nucleotide sequence previously allowed for the study of distance dependence, sequence dependence, and directionality of excess electron transfer reactions. This system was used here to determine if a polaron type mechanism was operative in excess electron transport in analogy to such observations in hole transfer. At least for the systems examined in this work, a polaron type mechanism does not appear operative for excess electron transfer. In order to determine if excess electron transport efficiency is dependent on the reduction potential of the aromatic amine, the redox potentials of a variety aromatic amine were determined. These aromatic amines differed by the addition of electron donating groups and π conjugation. Preliminary studies in this final model system with an aromatic amine of strong reduction potential showed no difference in excess electron transport when compared to the aromatic amine with a weaker reduction potential.