Molecular Studies of Cellular Response to the Genetic Defects of Thymidylate Biosynthesis in Saccharomyces cerevisiae

• Main Authors: Chia-Yi Chien, 簡佳怡
• Other Authors: Jin-Yuan Su
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/56824901067104772233

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 97 === The budding yeast Saccharomyces cerevisiae is considered one of the most ideal eukaryotic model systems in current biological studies. The elegance of yeast genetics and the development of modern molecular techniques in yeast allow its use for conveniently analyzing and dissecting gene functions in many biological aspects. Like in other eukaryotes, yeast possesses a defined genome, which is composed of 16 chromosomes, and is confined in a single nucleus in the haploid cell. The study of the mechanisms in maintaining the genomic integrity by using yeast as a model organism has gained a great deal of attention in recent years. This thesis is focusing on the study of how the regulation of DNA precursors synthesis would have any impact on the maintenance of genomic integrity in yeast. An adequate and proper supply of deoxyribonucleoside triphosphates (dNTPs) is crucial for DNA replication. Among the four dNTPs, the biosynthesis of thymidylate is distinct from the other three nucleotides. Namely, in the pyrimidine biosynthesis pathway, uridylate is the first synthesized precursor which is then converted to form thymidylate. Given the unique synthesis pathway of thymidylate, the study of the enzymes involved in this pathway has been an interesting subject in either clinical or biochemical research fields. This thesis has taken advantage of two essential genes whose products are required for the biosynthesis of thymidylate in yeast and analyzed their functions to address the question of how the defect of these genes would affect the growth of yeast from both cytoplasmic and nuclear perspectives. In eukaryotic cells, the biosynthesis of dNTPs occurs in the cytoplasm and is normally under tight control such that many enzymes involved in the synthesis of dNTPs are subject to cell cycle regulation. In the yeast, the genes encoding thymidylate synthetase (CDC21) and thymidylate kinase (CDC8) are both required for nuclear and mitochondrial DNA replication and the expression of these genes are cell cycle regulated. Early studies revealed that high frequency of respiratory-deficient petites (mitochondrial mutants) were formed in heat-sensitive cdc21 and cdc8 mutants grown at the permissive temperature. However, the molecular mechanism involved in such petite formation is largely unknown. A yeast cdc21-1 mutant was therefore used to demonstrate that the mutant cells accumulated dUMP in the mitochondrial genome as a result of the impaired thymidylate synthesis. We went on to prove that the initiation of Ung1p-mediated base excision repair in the uracil-laden mitochondrial genome in a cdc21-1 mutant is responsible for the cytoplasmic petite formation. Sexual conjugation in the budding yeast occurs between two haploid cells of opposite mating type. Conjugation commences with cytoplasmic fusion to form a zygote followed by fusion of the two parental nuclei to produce a diploid zygote. We discovered a distinctive feature that the haploid ung1Δ cdc21-1 double mutant cells lost the ability to mate with the UNG1+ cells. The nucleus of ung1Δ cdc21-1 appeared to be completely arrested upon the conjugation with UNG1+ cell. Zygotes formed under this circumstance were, as a result, unable to complete karyogamy (nuclear fusion) and perished. We illustrated in this thesis an exceptional DNA damage checkpoint function during yeast mating. Our studies have provided, for the first time, an ideal model to characterize the DNA damage checkpoint function occurring during cell fusion.