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Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 171-187). / Microfiche. / xix, 187 leaves, bound ill. 29 cm

<p>Eukaryotic Topoisomerase 2 is an essential enzyme that solves DNA topological problems such as DNA knotting, catenation, and supercoiling. It alters the DNA topology by introducing transient double strand break in one DNA duplex as a gate for the passage of another DNA duplex. Two different aspects of studies about eukaryotic Topoisomerase 2 will be covered in this thesis. In the first half of the thesis, we investigated conformational changes of human Topoisomerase 2&#61537; (hsTop2&#61537;) in the presence of cofactors and inhibitors. In the second half, we focused on an unknown regulatory function in the C-terminal domain (CTD) of Drosophila Topoisomerase 2 (Top2).</p><p>In the project of studying enzyme conformational changes, we adapted a previously developed methodology, Pulse-Alkylation Mass Spectrometry, with monobromobimane to study the protein dynamics of hsTop2&#61537;. Using this method, we captured the evidence of conformational changes in the presence of ATP and Mg2+ or the Top2 inhibitor, ICRF-193 which were not previously observed. Last, by using CTD truncated hsTop2&#61537;, the increasing reactivity of Cys427 suggested the CTD domain might be tethered adjacent to the core enzyme.</p><p>Following the study of enzyme conformational changes, we switched gear to examine an interaction between Drosophila Top2 and Mus101, homolog of human TopBP1. We first found that Mus101 interacts with CTD of Top2 in a phosphorylation-dependent manner. Next, in the co-immunoprecipitation and pull-down experiments using truncated or mutant Top2 with various Ser to Ala substitutions, we mapped the binding motif to the last amino acids of Top2 and identified that phosphorylation of Ser1428 and Ser1443 is important for Top2 to interact with the N-terminus of Mus101, which contains BRCT1/2 domains (BRCT, BRCA1 C-terminus). The binding affinity of the N-terminal Mus101 with a synthetic phosphorylated peptide covering the last 25 amino acids of Top2 (with pS1428 and pS1443) was determined by surface plasmon resonance with a Kd of 0.57 &#956;M. In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response to Mus101. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2&#61508;20, truncation of 20 amino acids from the C-terminus of Top2, developed abnormally high chromosome numbers, which implies an infidelity in chromosome segregation during mitosis. Lastly, Top2-null flies rescued by Top2 with S1428A and S1443A were found to be viable but sterile. After investigating spermatogenesis, telophase of meiosis I was delayed, indicating Top2-Mus101 interaction is also important in segregating DNA in meiosis.</p> / Dissertation

The genetic pathways influencing alcohol abuse and dependence are poorly characterized. Many critical discoveries about the interactions between ethanol-related behaviors and genetics have been made in the fruit fly Drosophila melanogaster. Coupling the statistical power of model organism studies to human association studies bolsters the analytical efficacy of these genomic approaches. A variety of behavioral assays are available for assessing behavioral responses to ethanol in Drosophila. However, we find our previously described eRING assay is influenced by the commonly used transgenic marker mini-white. We developed a Simple Sedation Assay (SSA) that is insensitive to the effects of white and mini-white. In SSAs, expression of endogenous wild-type white was not necessary for normal responses to ethanol. Neither expression nor RNAi-mediated knockdown of the transgenic mini-white influenced the effects of ethanol in flies. Critically, mini-white expression did not affect the phenotypes of flies with known alterations in ethanol sensitivity. Also, loss of function mutations in Clic show decreased sensitivity to ethanol in both eRING assays (as previously reported) and SSAs. Therefore, we explored the role of the known Clic interactors, TGF-β and ryanodine receptors. These studies were inconclusive but do not exclude the need for future work. Finally, using bioinformatic tools we constructed a mutli-species network of genes predicted to interact with Clic. Our RNAi screen against the Clic network serves as an important proof-of-concept and holds great potential for uncovering important therapeutic targets for alcohol use disorders.

Male Drosophila melanogaster double the transcription of most of the genes on their single X chromosome, to equal that from the two female X chromosomes, in a process termed dosage compensation. This process is mediated by the MSL complex, consisting of both protein and non-coding RNA components. This complex is only active in males due to the presence of MSL2, which is not translated in females. The X-linked Lsp1α gene of Drosophila melanogaster appears to escape dosage compensation, and exhibits two-fold higher levels of expression in females compared to males. The apparent lack of dosage compensation of Lsp1α could be due to the promoter being more active in females than in males, or to a lack of regulation by the MSL complex. In this study, the mechanism by which this happens has been addressed. Lsp1α is expressed exclusively in the fat body tissue of third instar larvae, and forms part of a multi-protein complex that acts as a nutrient reservoir during pupariation. In this study it has been shown that transgenes, in which the reporter gene, lacZ, is under the control of the Lsp1α promoter, exhibit variable levels of increased activity in female compared to male third instar larvae. At high levels of transgene expression, activity of the transgene is equal in female and male larvae. When the expression of the transgene is low, the activity of the transgene is much higher in female compared to male larvae. This increased sensitivity of the Lsp1α promoter to position effects in females appears to be mediated by one or more components of the MSL complex. Females ectopically expressing MSL2 exhibit decreased levels of transgene activity. Furthermore, overexpression of MSL1 causes an increase in the activity of transgenes subject to strong position effects. Despite these findings, the sex-specific regulation of the Lsp1α promoter does not account for the non-dosage compensated appearance of Lsp1α. Instead, unlike control dosage compensated X-linked genes, Lsp1α is not enriched for a histone modification, acetylation of lysine 16 of histone H4 that is essential for dosage compensation by the MSL complex. The developmental stage at which the four genes flanking Lsp1α are expressed has been determined using northern RNA hybridization. Expression of the gene immediately 3' of Lsp1α could not be detected at any developmental stage using northern RNA hybridization or in adults by RT-PCR. However, the two genes flanking Lsp1α are expressed in equal levels in male and female Drosophila as determined by quantitative RNase protection analysis. Furthermore, the regions between Lsp1α and these flanking dosage compensated genes do not prevent dosage compensation of an X-linked armlacZ reporter gene. Bioinformatic analysis shows that Lsp1α is present in three species closely related to D. melanogaster but is absent in more distantly related species. It is probable that because of its recent evolutionary origin, the Lsp1α gene lacks the DNA sequences that are required to attract the MSL complex. More generally, a model is proposed in which dosage compensation involves binding of the MSL complex to DNA sequences in actively transcribed regions with possible limited spreading to closely associated active genes.