<p>Since RNA Polymerase II (RNAPII) transcribes much of the genome, it is well situated to encounter and initiate a response to various types of DNA damage. However, to date very little is known about any response of RNAPII to DNA damage outside of Transcription Coupled Nucleotide Excision Repair (TC-NER). A link between DNA damage response mechanisms and the C-terminal domain of RNAPII (CTD) is suggested by an overlap between proteins that bind the CTD and genes required for resistance to DNA damaging agents. In this thesis, I show that proper deployment of CTD associated proteins is required to respond to DNA damaging agents. Furthermore, I show that a CTD associated protein (Set2) is required for response to DNA damage, but its catalytic activity is not. Finally, I show that the recombinational ability of strains lacking the CTD kinase, Ctk1, is deficient. Based on these lines of evidence, I propose a novel CTD Associated DNA Damage Response (CAR) system of proteins that is required for proper response to DNA damaging agents.</p> / Dissertation

Biochemistry Structural and Cholesterol Binding Properties of the Amyloid Precursor Protein C-Terminal Fragment C99 and the Etiology of Alzheimers Disease Paul John Barrett Dissertation under the direction of Professor Charles Sanders Alzheimers Disease (AD) is a severe form of dementia that currently affects nearly 40 million people worldwide, a number that is estimated to increase by the year 2050 to nearly 120 million. The production and oligomerization of the amyloid beta polypeptides (Aβ) is widely thought to play a central role in AD onset and progression. Aβ oligomers are neurotoxic, such that factors that increase Aβ production and propensity to oligomerize or that reduce its degradation and transport out of the brain are viewed as pro-AD risk factors. Recently, it has been established that cholesterol may be a pro-AD risk factor, but this mechanism is still highly controversial. Aβ polypeptides are derived from two step proteolytic processing of the amyloid precursor protein (APP) by the enzymes β- and γ-secretase. This thesis will investigate both how the structure of C99 and the ability of C99 to bind cholesterol regulate and promote these cleavage events. First, this thesis will focus on the three dimensional structure determination of C99 using NMR spectroscopy. Prior to this work, much had been inferred about how the structure of C99 regulated the cleavage events of the amyloidogenic pathway, but without an actual structure, this work was incomplete. I demonstrate that C99 contains a membrane embedded helical turn N-terminal of the transmembrane helix, and that the transmembrane helix is both highly curved and flexible in nature. We hypothesize from this work that the curved structure of the transmembrane helix allows for proper insertion in the γ-secretase complex, and may be a potential target for modulating Aβ production. Secondly, this thesis will focus on the mechanism that makes cholesterol a pro-AD risk factor. We have shown that not only can C99 specifically bind cholesterol, but that this binding event partitions C99 to cholesterol rich membrane domains. It is known that both β- and γ-secretase reside in these cholesterol rich membranes. These findings show that cholesterol binding by APP may be the first step in promoting Aβ formation during AD.

Activation of the p75 neurotrophin receptor leads to a variety of effects within the nervous system, including neuronal apoptosis. Both c-Jun N-terminal kinase (JNK) and the tumor suppressor p53 have been reported to be critical for this receptor to induce cell death; however, the mechanisms by which p75 activates these pathways is undetermined. I report that the Neurotrophin Receptor Interacting Factor (NRIF) is necessary for p75-dependent JNK activation and apoptosis. Upon NGF withdrawal, nrif -/- sympathetic neurons underwent apoptosis, while p75 mediated death was completely abrogated. The lack of cell death correlated with a lack of JNK activation in the nrif -/- neurons, suggesting that NRIF is a selective mediator for p75-dependent JNK activation and apoptosis. While I have shown NRIF to be essential for p75-induced cell death, there is still relatively little known about this protein. To this end, I demonstrate that NRIF is capable of inducing apoptosis in primary cells in the absence of p75 activation. This apoptotic program involves the activation of caspase 3 and the release of cytochrome c from the mitochondria. NRIF expression in primary cells is not sufficient to activate JNK but NRIF-activated cell death requires both p53 and its upstream activator p19Arf. NRIF is found in the nucleus after receptor activation, suggesting that this molecule may act as a transcription regulator. Consistent with this hypothesis, NRIF requires its putative DNA binding domain to maximally induce apoptosis. Finally, I examined the role of NRIF as a tumor suppressor and find that while this protein causes cell cycle arrest and apoptosis, the absence of nrif in vivo does not predispose mice to spontaneous tumor development. Taken together, these results establish NRIF as a proapoptotic protein that is essential for p75-mediated apoptosis.

The function of the human cardiac voltage-gated sodium channel hH1 is regulated in part by an EF-hand in its C-terminal cytoplasmic domain. The binding of calcium to the EF-hand results in calmodulin-independent, calcium-dependent changes in channel behavior. The channel is also regulated via an extrinsic calcium sensing pathway mediated by calmodulin (CaM), which binds to an IQ motif immediately adjacent to the EF-hand domain. Our results demonstrate the presence of these sensors and suggest that they are coupled through the IQ motif, which serves in this case as a molecular switch. Initially the IQ motif recruits CaM with strong affinity. When calcium levels are elevated, CaM binds calcium and undergoes a conformational change that reduces its affinity for the IQ motif. Release of the IQ motif would promote its interaction with the intrinsic calcium sensor, which raises the calcium affinity of the intrinsic sensor 1000-fold. Preliminary results suggest that Ca2+-loaded CaM binds to a remote site on hH1, supporting this hypothesized sequence of events. A comprehensive molecular mechanism for the Ca2+-dependent regulation of hH1 is proposed.

Topoisomerase II is an essential enzyme that controls DNA topology. This enzyme generates a protein-linked DNA break as a catalytic intermediate. This intermediate, known as the cleavage complex, is present in very low levels under normal conditions. Topoisomerase II poisons increase the levels of cleavage complexes and generate double-stranded DNA breaks that are mutagenic and can cause cell death. Abasic sites poison topoisomerase II-alpha. In this dissertation, the effect of exocyclic DNA adducts and abasic sites on enzyme-mediated DNA cleavage were examined. Endogenous DNA lesions such as etheno-dC, etheno-dG, and M1dG were strong topoisomerase II-alpha poisons. Studies of the mechanism by which these lesions poison the enzyme revealed that these lesions did not inhibit enzyme-mediated religation, and that the enzyme did not posses higher affinity for these adducted DNA substrates. These findings suggest that the presence of DNA lesions accelerated the forward rate of topoisomerase II-DNA cleavage complex formation at the chemical scission step. A fluorescence resonance energy transfer system showed a correlation between DNA bending induced by different lesions and the ability of topoisomerase II-alpha to cleave the DNA substrate. Moreover, human cells treated with the alkylating agent 2-chloroacetaldehyde, which induces etheno adducts, displayed increased levels of topoisomerase II-alpha-associated DNA breaks. Furthermore, cells treated with the methylating agent methyl methanesulfonate (MMS) displayed increased levels of enzyme-associated DNA breaks with a parallel increase in the amount of abasic sites. Methylated bases are poor topoisomerase II poisons, but are readily converted to abasic sites by N-methyl purine glycosylase (MPG). In addition, cells overexpressing MPG displayed higher baseline levels of topoisomerase II-alpha-associated DNA breaks. These data suggest that topoisomerase II-alpha interacts with intermediates of the base excision repair pathway. Finally, cells with decreased levels of topoisomerase II-alpha displayed a 40% reduction in the amount of double-stranded DNA breaks generated upon MMS treatment, and displayed a slight resistance to MMS cytotoxicity. These findings demonstrate that topoisomerase II-alpha is able to interact with DNA lesions in a physiological system and that the enzyme mediates a portion of the clastogenic and cytotoxic effects of MMS.

HUMAN TOPOISOMERASES AND DNA GEOMETRY: PUTTING A POSITIVE TWIST ON ENZYME ACTION AMY KATHLEEN MCCLENDON Topoisomerases play critical roles in maintaining DNA topology during cellular processes such as DNA replication in eukaryotes. Movement of the replication machinery through the double helix induces positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single- and double-stranded DNA breaks, respectively, it has been assumed that topoisomerase I relaxes the positive supercoils while topoisomerase II resolves precatenanes. In contrast to this proposed segregation of function, models for anticancer drug action place topoisomerase II ahead of replication forks. This discrepancy raises the question of whether eukaryotic type II topoisomerases have normal physiological functions ahead of DNA tracking systems. If so, then positively supercoiled DNA might be the preferred substrate for human topoisomerase II?, the isoform involved in replicative processes. Therefore, the work described in this dissertation compared the activities of human topoisomerases on positively and negatively supercoiled DNA in the absence and presence of anticancer drugs, and explored the mechanisms by which topoisomerase II recognizes DNA supercoil geometry. First, this work characterized the abilities of human topoisomerase II? and ? to relax positively and negatively supercoiled DNA. Topoisomerase II?, but not ?, displayed characteristics that suggest it has the potential to relieve torsional stress ahead of approaching DNA tracking systems efficiently and safely. Second, this work examined the effects of positive DNA supercoiling on topoisomerase-mediated DNA cleavage and response to anticancer agents. Results indicate that DNA supercoil geometry has a profound influence on topoisomerase II-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either type II enzyme. Lastly, this work explored the mechanism by which topoisomerase II recognizes DNA supercoil geometry. Results suggest that the enzyme recognizes supercoil geometry in a bimodal fashion that involves elements in the N-terminal or central domain for cleavage and the variable C-terminal domain for relaxation. This ability has implications for the catalytic function of topoisomerase II and may account for some of the differences in the physiological roles played by distinct type II enzymes.

The objective of this research was to examine the FosX class of metalloenzymes utilized by pathogenic microorganisms for resistance to the antibiotic fosfomycin. Fosfomycin is an extremely stable natural product produced by strains of soil-dwelling Streptomyces and possesses desirable pharmacological properties. Unfortunately, the existence of fosfomycin-inactivating enzymes endangers the clinical value of this drug. Present research focuses on mechanistic and structural characterization of fosfomycin-inactivating enzymes from the FosA, FosB, and FosX classes of fosfomycin resistance proteins. The well characterized FosA proteins possess robust catalytic activity while the FosB and FosX proteins show significantly less catalytic ability. The FosA and FosB enzymes are thiol-transferases that inactivate fosfomycin through conjugation with glutathione and cysteine, respectively. The more distantly related enzymes of the FosX class, however, catalyze the hydrolysis of fosfomycin. Comparison of FosX proteins from diverse microorganisms reveals a range of catalytic activity, catalytic promiscuity, and ability to confer resistance to fosfomycin in a model organism (E. coli). Critical issues that were addressed in this research included determination of enzyme activity both in vitro and in the biological setting of E. coli, determination of metal binding kinetics, and elucidation of catalytically important resdiues through site-directed mutagenesis studies. Although it is beyond the capacity of this research, elucidation of the molecular basis of resistance will aid the development of inhibitors for these antibiotic-inactivating enzymes.

This project involved the investigation of both the dynamic features of the dimer interface of the Mu class GSH transferase rGSTM1-1, as well as, the structural and mechanistic characteristics of HCCA Isomerase, an enzyme related to the mitochondrial (Kappa) GSH transferase family. The dynamics along the dimer interface of rGSTM1-1 were probed by using site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry. This work led to hypotheses about the roles of both hydrophobic and electrostatic motifs along the interface. It also resulted in more clear understanding of the regions within each monomer that are important for the stability of individual subunits. Crystallography of HCCA Isomerase in conjunction with activity assays on the native substrates allowed us to conclude that this enzyme is definitely a Kappa GSH transferase. Transient state kinetic measurements with native substrates and analogs as well as crystallography with one of the analogs allowed us to propose a global pathway for HCCA Isomerases catalytic mechanism.

Topoisomerases play critical roles in maintaining DNA topology during cellular processes such as DNA replication in eukaryotes. Movement of the replication machinery through the double helix induces positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single- and double-stranded DNA breaks, respectively, it has been assumed that topoisomerase I relaxes the positive supercoils while topoisomerase II resolves precatenanes. In contrast to this proposed segregation of function, models for anticancer drug action place topoisomerase II ahead of replication forks. This discrepancy raises the question of whether eukaryotic type II topoisomerases have normal physiological functions ahead of DNA tracking systems. If so, then positively supercoiled DNA might be the preferred substrate for human topoisomerase IIá, the isoform involved in replicative processes. Therefore, the work described in this dissertation compared the activities of human topoisomerases on positively and negatively supercoiled DNA in the absence and presence of anticancer drugs, and explored the mechanisms by which topoisomerase II recognizes DNA supercoil geometry. First, this work characterized the abilities of human topoisomerase IIá and â to relax positively and negatively supercoiled DNA. Topoisomerase IIá, but not â, displayed characteristics that suggest it has the potential to relieve torsional stress ahead of approaching DNA tracking systems efficiently and safely. Second, this work examined the effects of positive DNA supercoiling on topoisomerase-mediated DNA cleavage and response to anticancer agents. Results indicate that DNA supercoil geometry has a profound influence on topoisomerase II-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either type II enzyme. Lastly, this work explored the mechanism by which topoisomerase II recognizes DNA supercoil geometry. Results suggest that the enzyme recognizes supercoil geometry in a bimodal fashion that involves elements in the N-terminal or central domain for cleavage and the variable C-terminal domain for relaxation. This ability has implications for the catalytic function of topoisomerase II and may account for some of the differences in the physiological roles played by distinct type II enzymes.

The objective of this research was to investigate the structure and catalytic mechanisms of the FosX protein class. These enzymes are epoxide hydrolases that disrupt the first step of bacterial cell wall biosynthesis, thereby inactivating the antibiotic fosfomycin. Data presented in this work concerns FosX enzymes from the genome of pathogenic microorganism Listeria monocytogenes and a recently discovered integron isolated from Pseudomonas putida. Hydrogen-deuterium exchange mass spectrometry was used to determine structural perturbations that occur when the Listeria protein binds to divalent metal species. In addition, steady-state metal binding to Listeria FosX was investigated using optical spectroscopy. Finally, the putative FosX gene from Pseudomonas was cloned and the protein product expressed and purified. The enzyme was tested for FosX activity and ability to confer fosfomycin resistance. The ultimate goal of this research is to elucidate further mechanistic details about this class of fosfomycin resistance proteins, with the hope to one day develop inhibitors that will lessen or eliminate bacterial resistance to this antibiotic.