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Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 90-99).

Msh2 and Mlh1 proteins from Tetrahymena thermophla are homologues of MutS and MutL from Escherichia coli respectively. MutS and MutL are DNA mismatch repair proteins. In eukaryotes, MutS homologues recognize the replication errors and MutL homologues interact with MutS homologues and other proteins to make the repair occur. Biolistic transformation has been done to make the msh2 and mlh1 single knockouts in the macronuclei of different strains and the knockouts were verified complete. Two strains of WT crossing KO or KO crossing KO, with different mating types, were induced to conjugate. The processes were studied by microscopy using DAPI staining. For the msh2 knockouts, there were no crescent micronuclei formed throughout the conjugation of two knockout cells, and the pairing level was reduced severely. However, a knockout cell and a wild-type cell could conjugate normally at a high level pairing efficiency. Msh2 protein seems to be important to cell pairing and indispensible for the formation of the crescent micronuclei during cell conjugation. For the mlh1 knockouts, the pairing level of a knockout and a wild-type was reduced by half and the pairing level of two knockouts was reduced more than 80%; however, the paired cells in both could complete the conjugation with delay. Pms2 protein may have redundant roles in the MutL heterodimer (Mlh1-Pms2). In addition, chemical mutagens treated knockout was crossed with non-treated wild-type and the conjugation was compared with treated wild-types. Most of the treated knockout cells could not pair after starvation and mixing with non-treated wild-type cells, which means most of the cells could not enter meiotic phase. It is probable that G2/M checkpoint arrested the meiotic cell cycle and the intra-S phase was inactivated. Thus, Msh2 protein may have a role in the meiotic intra-S phase checkpoint system.

The DNA of all organisms is continuously exposed to exogenous and endogenous genotoxic agents. Fortunately, through the concerted actions of several DNA repair and mutation avoidance pathways, DNA damage can be removed and an organism’s genomic stability maintained. DNA base-base mismatches are generated as a result of the inherent replication errors made by the DNA replication machinery, as well as during the meiotic pairing of homologous but non-identical chromosomes. Through the coordinated actions of the highly conserved DNA mismatch repair (MMR) system, these errors are detected, removed and corrected, thus restoring the integrity of the DNA. In the absence of DNA MMR, genetic instability is unavoidable, resulting in the accumulation of mutations, and in mammals, a susceptibility to cancer. To better understand the roles of the MMR system in mutation avoidance during DNA replication, meiosis, and in nuclear apoptosis, we have utilized the nuclear dimorphic, ciliate protozoan Tetrahymena thermophila. We have identified seven putative MMR homologues; two are similar to eukaryotic MLH1 and PMS2, respectively, and five are similar to eukaryotic MutS homologues, one with eukaryotic MSH2 and four with MSH6. Our studies demonstrate that during conjugation, the relative transcript abundance of each MMR homologue is increased compared to vegetatively growing or nutritionally deprived (starved) cells. Also, the expression profile throughout conjugation is bimodal, corresponding to micronuclear (MIC) meiosis and macronuclear (MAC) anlagen development, both periods in which DNA replication occurs. Cells containing macronuclear knockouts of the PMS2, MSH2 and MSH6_1 genes were unable to successfully pair and complete conjugation, but were viable throughout vegetative growth. Cells in which the macronuclear MSH6_2 gene was knocked out had a phenotype that was similar to wild-type cells, during conjugation and vegetative growth. Interestingly, we observed that the MIC of cells containing MAC knockouts of the PMS2 and TML1 genes appear to have decreased copy number of specific “target sequences”, as determined by qPCR using the Random Mutation Capture (RMC) assay. This decrease reflects neither a loss of micronuclei nor a reduction in total micronuclear DNA content. These studies demonstrate that the PMS2, TML1, MSH2, and MSH6_1 homologues are necessary for the maintenance of micronuclear function and stability during conjugal development and vegetative growth, whereas the remaining MSH6 homologues have less pronounced roles in DNA repair and development. Additionally, macronuclear development in Tetrahymena appears less reliant on the DNA mismatch repair system and perhaps uses alternate surveillance mechanisms to maintain genomic stability during asexual and sexual development. / Graduate / 0306 / 0379 / 0307

DNA mismatch repair (MMR) is an essential part of genomic stability, guarding the integrity of the genome in virtually all cells. MMR corrects mismatched bases in DNA and is one of several DNA repair pathways conserved from bacteria to humans. In Escherichia coli, MutS and MutL are the key proteins in MMR. In prokaryotes, the MMR proteins function as homodimers whereas in eukaryotes the MMR proteins come together as heterodimers. In the absence of MMR there is a great increase in mutation frequency and a higher susceptibility to cancer in mammals. Previous studies in our laboratory have identified five MutS homologs and two MutL homologs in the single-celled ciliated protozoan, Tetrahymena thermophila. MMR repair has been extensively studied in E. coli but less is known in eukaryotes. T. thermophila 's biology and the recent sequencing of its genome make it an attractive eukaryotic research model. In this study, poly-histidine tagged MMR genes from T. thermophila and human thymine DNA glycosylase (TDG) were cloned into two different types of T thermophila expression plasmid. The integrated homologously recombinational T. thermophila vector approach and the espisomal rDNA vector approach which utilized Gateway® technology a cloning method based on the bacteriophage lambda site-specific recombination system. The homologous vector approach relies on mutant strains of T. thermophila harboring a negatively selectable allele of a P-tubulin gene producing sensitivity to paclitaxol. Upon knocking out the mutant J3-tubulin with the gene of interest the resistance of the Tetrahymena strain is then resorted and selected for. The various T. thermophila expression plasmids and DNA transformations techniques to follow such as biolistic bombardment and conjugated electroporation were carried out in order to optimize the technical aspect of working with T. thermophila. and finally lead to the purification of histidine tagged T. thermophila MMR proteins for antibody production and further studies. This study will lead to insight into the inner workings of DNA mismatch repair and technical aspect of working with the organism and will permit functional and structural studies of the MMR homolog proteins in T. thermophila.

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