Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 74-87).

Note:

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 104-113).

DNA double strand breaks (DSB) are the most toxic of all types of DNA lesions. In Saccharomyces cerevisiae, DNA DSBs are predominantly repaired by the homologous recombination repair (HRR) pathway. The initial step of HRR requires extensive processing of DNA ends from the 5' to 3' direction by specific endo-exonuclease(s) (EE) at the DSB sites, but no endo-exonuclease(s) has yet been conclusively determined for such processing of DSBs. S. cerevisiae TRM2/RNC1 is a candidate endo-exonuclease that was previously implicated for its role in the HRR pathway and was also shown to have methyl transferase activity primarily located at its c-terminus. / In this dissertation, we provided compelling biochemical and genetic evidence that linked TRM2/RNC1 to the DNA end processing role in HRR. Trm2/Rnc1p purified with a small calmodulin binding peptide (CBP) tag displayed single strand (ss) specific endonuclease and double strand (ds) specific 5' to 3' exonuclease activity characteristic of endo-exonucleases involved in HRR. Intriguingly, purified Trm2/Rnc1p deleted for its C-terminal methyl transferase domain retained its nuclease activity but not the methyl transferase activity indicating that the C-terminal part responsible for its methyl transferase function is not required for its nuclease activity. / Our genetic and functional studies with S. cerevisiae trm2/rnc1 single mutants alone or in combination with other DNA DSB repair mutants after treatment with the DNA damaging drug methyl methane sulfonate (MMS) or IR that is believed to produce DSBs, or with specific induction of DNA DSBs at the MAT locus by HO-endonuclease demonstrated an epistatic relationship of TRM2/RNC1 with the major recombination factor RAD52. These studies suggested that TRM2/RNC1 probably acts at an earlier step than RAD52 in the HRR pathway. The genetic evidence also indicated a possible functional redundancy with the bona fide endo-exonuclease EXO1 in the processing of DNA ends at the DSB sites. / In a recent report, the immuno-purified mouse homologue of TRM2/RNC1 exhibited similar enzymatic properties as the endo-exonucleases involved in HRR. A small molecular inhibitor pentamidine specifically inhibited the nuclease activity of the mouse EE and sensitized various cancer cells to DNA damaging agents commonly used in cancer chemotherapy. We specifically suppressed expression of the mouse EE using small interfering RNA (siRNA) that conferred sensitivity of B16F10 melanoma cells to a variety of DNA damaging drugs often used in cancer treatment. This further validated our earlier claim of the endo-exonuclease as a potential therapeutic target in treating cancer.

The ability to evade apoptosis is an acquired characteristic associated with many normal and pathophysiological processes. TMEM 85 represents a novel transmembrane domain containing human protein isolated in our previous screen for Bax suppressors, but whose function is currently unknown. Using viability and growth assays, we confirmed that TMEM 85 is anti-apoptotic. Four unique human cDNA sequences containing regions distinct from and of perfect identity to our cDNA were present in the database. Analysis of TMEM 85 suggests that it consists of five exons, alternatively spliced to produce at least four different mRNA's and proteins (TMEM 85v1-v4). RT-PCR analysis using RNA isolated from mice and humane tissues show that all transcripts are expressed. Yeast contain an orthologue of the human TMEM 85v1 protein, YGL213C. Surprisingly, the viability assay indicated that mutants lacking YGL231c do not show a hyper-responsive apoptotic phenotype, however its overexpression shows that it is nevertheless anti-apoptotic. Using a yeast strain expressing chromosomally TAP-tagged YGL231c, we found no up-regulation of the endogenous gene due to stress. The deletion mutant is also known to expresses a synthetically lethal phenotype in the presence of alpha-synuclein. While expression of alpha-synuclein caused significant death in both the wild type and deletion mutants, TMEM 85v2 was unable to exhibit a protective role. These findings demonstrate the complexity of the TMEM 85 gene and its anti-apoptotic function in both yeast and human.

Saccharomyces cerevisiae has developed mechanisms in order to survive harsh environmental conditions. This species responds to stresses such as ethanol, heat, and weak acid exposure via two well-characterized stress response pathways. These typically involve either the Hsf1p or the Msn2/4p transcriptional regulators. Recently, our lab has begun to characterize a member of the zinc cluster protein family: Asg1p (Activator of Stress Genes, systematic name: YIL130W), which is presumed to stimulate stress response genes independently of the Hsflp and Msn2/4p pathways. Previous work has revealed five target genes of Asg1p (HSP30, STP4, YER130C, TPO2, YRO2) thought to be involved in this novel stress response pathway. In this study, we attempted to better characterize the role of Asg1p and its target genes during stress induction. We first determined if the induction of certain Asg1p target genes by stress is strain specific. HSP30 induction by heat shock is specific to the W303 strain as shown by primer extension analysis. We then generated the deletion strains Deltaasg1 and Astp4 in W303. We observed a loss of induction of HSP30 in the Deltaasg1 deletion strain when cells were exposed to ethanol. This led us to believe that Asg1p does play a role in the stress response pathway. Also, we attempted to globally define the target sites of Asg1p in vivo on a genome-wide scale by combining Chromatin Immuno Precipitation with microarrays (ChIP-chip). We identified eight putative Asg1p target genes: YRO2, HSP78, ZRT2, ZRT1, MSN4, STP4, TPO2, and HSP30.