Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 117-122).

In order to maintain genome stability, DNA damage needs to be detected and repaired in a timely fashion. To cope with damaged DNA, cells have evolved mechanisms termed "checkpoints", where, upon damage, cells initiate a signal transduction cascade that results in the slowing or halting of the cell cycle, allowing efficient DNA repair. Defects in the DNA damage checkpoint result in an overall increase in genomic instability and are thought to fuel cancer progression. To facilitate our understanding of how DNA damage leads to cancer progression, it is crucial to fully comprehend how these signal transduction mechanisms function. In this work, we have characterized in great detail the mechanisms of regulation of Rad53 (a central regulator of the DNA damage response in Saccharomyces cerevisiae) at the genetic, biochemical and structural level. Firstly, we describe a complex biochemical two-step mode of activation of Rad53 by protein-protein interaction and multi-step phosphorylation. We also shed light onto the mechanisms by which Rad53 is turned off to allow the cell cycle to resume, a process termed DNA damage recovery and adaptation. We found that during adaptation, the polo-like kinase Cdc5 is required to attenuate Rad53 catalytic activity. Finally, the study of Rad53 at the molecular and atomic level revealed that in addition to being regulated through a complex network of protein-protein interactions, Rad53 autophosphorylation is orchestrated by a mechanism of dimerization, activation segment phosphorylation via A-loop exchange, as well as through an autoinhibition mechanism regulated by a specific alpha-helical region at the C-terminal extremity of its kinase domain. Such work is important in understanding the function of different proteins in DNA damage signaling. This knowledge will enhance our understanding of the progression of DNA damage related diseases such as cancer, and could eventually help in the long term the development of novel therapeutics as treatments against these conditions.

The efficiency of cellulose hydrolysis under straight saccharification and simultaneous saccharification and fermentation (SSF) conditions was evaluated using three lignocellulosic materials (switchgrass, cornstover, and poplar), which had been pretreated with dilute sulfuric acid under conditions which optimized xylose concentrations in the prehydrolysate liquid. Yields of glucose, cellobiose and ethanol obtained from the pretreated feedstocks were measured over 168 hrs. The final theoretical conversions of cellulose from pretreated switchgrass, cornstover, and poplar in straight saccharification were 85-100% (average 94%), 84-100% (average 96%), and 75-100% (average 87%), respectively, while in SSF the conversions were 84-90% (average 87%), 91-96% (average 90%), 72%-82% (average 76%), respectively. The conversion rates of poplar in straight saccharification and SSF were significantly lower than those of switchgrass and cornstover. The effects of reaction parameters such as enzyme activity, cellulose availability, and yeast cell viability on the extent of hydrolysis in straight saccharification and SSF were also studied. Results indicate that the lower glucose or ethanol yields associated with some of the poplar were due to the recalcitrant nature of its cellulose. To compare accurately the efficiencies between straight saccharification and SSF, a direct method for determining the cellulose content of the feedstocks residues resulting from SSF experiments has been developed and evaluated. The method improves on classical cellulose assays by incorporating a yeast lysing enzyme to remove yeast glucans from the feedstocks residue prior to acid hydrolysis and subsequent quantification of cellulose derived glucose. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzyme lysis. The method was applied to the analysis of the cellulose and yeast-glucan content of SSF residues from the three pretreated feedstocks. Cellulose assays employing the lysing enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucan were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to the subsequent chemical and physical analysis of SSF residues. / Graduation date: 1996

Sulfite mutants representing five complementation groups, previously derived from an ethyl methanesulfonate-treated haploid strain of Saccharomyces cerevisiae were studied. Although the wildtype S. cerevisiae strain used (isogenic to X2180-1 A) had a basal tolerance for sulfite (7 μM free H₂SO₃), the sensitive and resistant mutants were found to tolerate less than 3 to 5.5, or greater than 19 μM free H₂SO₃, respectively. No apparent correlation was found between the response to sulfite and generation time in rich (YEPD) or minimal media. Resistant mutant 11-1 had an extended lag phase relative to wildtype. Mutant and wildtype proteins were labeled with ³⁵S-methionine to determine differences in banding patterns due to sulfite-specific induction or disappearance of polypeptides. No obvious differences following SDS-PAGE and autoradiography were observed upon induction with 0.213 μM free H₂SO₃. No consistent correlations were found between the sulfite phenotypes and responses to other reducing agents. Sensitive mutant 35-2 appeared to be three to ten times more sensitive to dithiothreitol than wildtype and sensitive mutant 47-9 was three to four times more sensitive to sodium nitrite and three to seven times more sensitive to sodium thiosulfate than wildtype. Log phase cells of sensitive mutant 33-2 were found to have significantly less glutathione than wildtype. Wildtype contained 62.6 nmol min⁻¹ mg protein⁻¹ (62.6 mU mg protein⁻¹) glutathione reductase (GR) and 2.78 nmol min⁻¹ mg protein⁻¹ (2.78 mU mg protein⁻¹) glutathione S-transferase (GST). Log phase cells of one resistant mutant showed a significantly higher level of GR than wildtype, 135%. The resistant mutants as well as some of the sensitive mutants had reduced GST levels. Survival rates of the mutants in buffer in the presence of sulfite did not correlate with their sensitive or resistant phenotypes, suggesting that survival and growth in the presence of sulfite are not necessarily related functions. Relative to wildtype, survival upon prolonged storage at 4°C was markedly reduced for two of the four sensitive mutants, one of which was 33-2, and was enhanced for one resistant and another sensitive mutant. / Graduation date: 1992

The global gene expression profiles of industrial strains of <i>Saccharomyces cerevisiae</i> responding to nitrogen deficiency and very high sugar concentrations stresses were determined by oligonucleotide microarray analysis of ~ 6200 yeast open reading frames. Genomics analysis showed that 400 genes in S. cerevisiae was differentially expressed by more than 1.5-fold compared with controls at late-logarithmic phase of fermentation, as the yeast adapted to changing nutritional, environmental and physiological conditions. The genes of many pathways are regulated in a highly coordinated manner. The repressed expression of GDH1 and up-regulation of ARO10 within the contrast of Q270/Q10 indicated high energy demanding of yeast cells under high sugar stress. Activities of G3P shuttle indicated that under very high gravity environment, sufficient assimilatory nitrogen enhances yeasts ability of redox balancing, and therefore higher stress-tolerance and higher fermentation efficiency of yeast. Under contrast W270/Q270, the up-regulation of DUR1,2 responsible for urea degradation induces the glutamate biosynthesis and the consumption of -ketoglutarate. This may indicate that higher nitrogen level would enable higher activities in the TCA cycle, and therefore generate more energy for biosynthesis and yeast cell proliferation under very high gravity fermentation conditions. Nitrogen metabolism was also stimulated by high nitrogen level when yeast was grown in very high gravity environment.

In order to maintain genome stability, DNA damage needs to be detected and repaired in a timely fashion. To cope with damaged DNA, cells have evolved mechanisms termed "checkpoints", where, upon damage, cells initiate a signal transduction cascade that results in the slowing or halting of the cell cycle, allowing efficient DNA repair. Defects in the DNA damage checkpoint result in an overall increase in genomic instability and are thought to fuel cancer progression. To facilitate our understanding of how DNA damage leads to cancer progression, it is crucial to fully comprehend how these signal transduction mechanisms function. In this work, we have characterized in great detail the mechanisms of regulation of Rad53 (a central regulator of the DNA damage response in Saccharomyces cerevisiae) at the genetic, biochemical and structural level. Firstly, we describe a complex biochemical two-step mode of activation of Rad53 by protein-protein interaction and multi-step phosphorylation. We also shed light onto the mechanisms by which Rad53 is turned off to allow the cell cycle to resume, a process termed DNA damage recovery and adaptation. We found that during adaptation, the polo-like kinase Cdc5 is required to attenuate Rad53 catalytic activity. Finally, the study of Rad53 at the molecular and atomic level revealed that in addition to being regulated through a complex network of protein-protein interactions, Rad53 autophosphorylation is orchestrated by a mechanism of dimerization, activation segment phosphorylation via A-loop exchange, as well as through an autoinhibition mechanism regulated by a specific alpha-helical region at the C-terminal extremity of its kinase domain. Such work is important in understanding the function of different proteins in DNA damage signaling. This knowledge will enhance our understanding of the progression of DNA damage related diseases such as cancer, and could eventually help in the long term the development of novel therapeutics as treatments against these conditions.

ABSTRACT The molecular mechanisms controlling longevity have been subject to intense scrutiny in recent years. It is clear that genomic stability, stress response and nutrient signaling all play critical roles in lifespan determination, but the precise molecular mechanisms and their often subtle influence on cellular function remain largely unknown. The Anaphase Promoting Complex (APC) is an evolutionarily conserved ubiquitin-protein ligase composed of 13 subunits in yeast, required for M and G1 cell cycle progression, and is associated with cancer and premature aging in many model systems when defective. The APC targets substrates for proteasome-dependent degradation, yet the full range of APC substrates and their role in mediating genomic stability, stress response and longevity are largely unknown. In this study, we use the model organism Saccharomyces cerevisiae to investigate the results of two screens designed to identify novel APC targets, regulators and/or modifiers, in an effort to better understand the function of the APC. Both of these screens made use of the Apc5 subunit. This subunit is likely an important structural component of the APC and may be targeted by many APC regulatory enzymes. This subunit is essential, but a temperature sensitive (ts) allele of Apc5 was available for these studies. First, a Yeast 2-Hybrid (Y2H) screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as a potential APC substrate. We hypothesized that the APC targets Fob1 for proteasome- and ubiquitin-dependent degradation. Authenticating Fob1 as a novel APC substrate makes up the first part of this thesis. We have found that Fob1 is unstable specifically in G1, and cycles throughout the cell cycle in a manner similar to Clb2, an APC target. Consistent with the APC mediating Fob1 degradation, Fob1 is stabilized in APC and proteasome mutants. Disruption of FOB1 in WT cells increased replicative lifespan, a measure of how many daughter cells a single mother will produce prior to senescence; moreover, FOB1 disruption improved APC mutant replicative lifespan defects. Increased FOB1 expression decreased replicative lifespan in WT cells, while increased expression in APC mutant cells did not reduce replicative lifespan further, suggesting an epistatic interaction. FOB1 deletion also suppressed cell cycle progression, and rDNA recombination defects observed in apc5CA cells. Mutation to a putative Destruction Box-like motif (Fob1E420V) disrupted Fob1 modification, stabilized the protein and increased rDNA recombination. These results support our hypothesis that Fob1 is a novel APC target and that Fob1 dosage may be regulated by the APC in response to cell cycle and environmental cues to regulate APC-dependent genomic stability and longevity. Second, an aptamer (small peptide) based screen identified peptides capable of suppressing the ts defect of the apc5CA mutant. One aptamer of interest is Y65, which has homology to the ubiquitin ligase Elc1. A Y2H found that this peptide Y65 binds the unstable stress response transcription factor Cin5. We hypothesized that this peptide may stabilize Cin5 by masking ubiquitin-dependent degradation. Stabilized Cin5 may in turn alleviate some apc5CA mutant defects. Characterizing Cin5 and confirming that Cin5 is subject to proteasome and ubiquitin-dependent degradation makes up the second portion of this thesis. During our investigation of Cin5 we identify a phospho-inhibited degradation motif within Cin5 that prevents ubiquitination and subsequent degradation when phosphorylated. We also provide evidence suggesting Cin5 may be targeted by a previously unidentified ubiquitin ligase subcomplex including Elc1 and Grr1. These data have helped elucidate the ubiquitin dependent regulation of Cin5. In summary, this research demonstrates the feasibility of using the Y2H and aptamer screens to identify and characterize molecular networks that interplay with the APC. Additionally, identifying and characterizing proteins where APC activity or function can be modified by aptamer binding has the potential to classify drug targets for therapeutic use in higher eukaryotes. Further understanding of the role the APC plays in cell cycle progression, chromatin assembly, genomic stability, stress response and longevity will be valuable to fundamental biological science, and may also have applications in health science and medicine.

The global gene expression profiles of industrial strains of <i>Saccharomyces cerevisiae</i> responding to nitrogen deficiency and very high sugar concentrations stresses were determined by oligonucleotide microarray analysis of ~ 6200 yeast open reading frames. Genomics analysis showed that 400 genes in S. cerevisiae was differentially expressed by more than 1.5-fold compared with controls at late-logarithmic phase of fermentation, as the yeast adapted to changing nutritional, environmental and physiological conditions. The genes of many pathways are regulated in a highly coordinated manner. The repressed expression of GDH1 and up-regulation of ARO10 within the contrast of Q270/Q10 indicated high energy demanding of yeast cells under high sugar stress. Activities of G3P shuttle indicated that under very high gravity environment, sufficient assimilatory nitrogen enhances yeasts ability of redox balancing, and therefore higher stress-tolerance and higher fermentation efficiency of yeast. Under contrast W270/Q270, the up-regulation of DUR1,2 responsible for urea degradation induces the glutamate biosynthesis and the consumption of -ketoglutarate. This may indicate that higher nitrogen level would enable higher activities in the TCA cycle, and therefore generate more energy for biosynthesis and yeast cell proliferation under very high gravity fermentation conditions. Nitrogen metabolism was also stimulated by high nitrogen level when yeast was grown in very high gravity environment.

In this thesis, the main objective is to perform statistical analysis of parameters yeast cell cycle regulated genes. We have known that there are 800 cell cycle regulated genes from Spellman et al. [9] (Spellman¡¦s 800) and 687 cell cycle regulated genes from MIPS database (MIPS¡¦s 687). We analyze yeast cell cycle regulated genes with statistical methods and models. The four main index statistics considered are as follow: 1. numbers of triscription factors bind to promoters, 2. coherence of genes with 104 known regulated genes (KRG) in alpha-factor experiment, 3. coherence of genes with 104 KRG in cdc15 experiment, 4. coherence of genes with 104 KRG in cdc28 experiment. Although, binding numbers can be fitted to geometric distribution in two subgroups of genes, we found that it is infeasible to classify the cell cycle regulated genes by only using HMM, and the coherence method also improve Spellman classification results if the standard is MIPS database or the 104 KRG. Finally, the cell cycle classification criterion in MIPS¡¦s 687 genes are found to include the information given by Spellman¡¦s 800.

YDL100Cp is the ArsA homologue protein found in S. cerevisiae. In bacteria, ArsA protein is involved in As3+detoxification but the function of YDL100Cp is still unknown. Previous studies show that deletion of YDL100C in S. cerevisiae was not lethal and had no effect on As3+ sensitivity or growth at 30¢J. However, when grown at 40¢J, growth of YDL100C disrupted strain (JSY1) was inhibited. To study the role of YDL100C in response to lethal heat shock, wild type (W303-1B) and JSY1 cells were exposed to 50¢J for 15 min. The survival rate of JSY1 cells was half of W303-1B cells and the difference in survival rate was complemented by introduction of plasmid carrying YDL100C. It suggests that YDL100Cp plays a role in acquisition of thermotolerance to lethal heat shock. It is believed that there are two factors involved in heat-induced cell death: the heat damage and the oxidative damage. Determinations of heat-damage related defense system in S. cerevisiae, including trehalose (a thermoprotectant) content, Hsp70 expression and Hsp104 expression, demonstrate that heat damage should not be the major cause of JSY1 cell death during heat shock. For the oxidative damage, the measurement of in vivo reactive oxygen species reveal the lower protein damage caused by reactive oxygen species (ROS) in JSY-1 after 50¢J 15 min heat shock, this might reflect the difference in viability of three strains under lethal heat shock. And with the intra cellular content of glutathione, it revels that the YDL100C deficient caused cell got more serious oxidative damage under 50¢J heat shock. But the observation of thermotolerance related ROS scavenger system (including the catalase, and superoxide dismutase) expression with reverse transcription polymerase chain reaction suggested that YDL100C deficient had no effect on triggering these system. As the result, it is suggested that the function of YDL100Cp in S. cerevisiae might be an oxidative damage repair system, such as the glutathione peroxidase. It might react with the oxidative damage substance and function as a deoxidizer.