Role of the ribosomal DNA repeats on chromosome segregation of Saccharomyces cerevisiae : a dissertation presented in fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand

Abstract

Chromosome segregation is a highly conserved process that progresses with great accuracy. Failure of proper segregation can lead to genetic disorders, such as Down syndrome in humans. Interestingly, segregation errors found in human genetic disorders and associated with spontaneous abortions or stillbirths are frequent in the chromosomes containing the ribosomal RNA gene repeats (rDNA). The rDNA is essential for cell viability and growth as it encodes ribosomal RNA, a major component of ribosomes. In yeast, the rDNA locus has a unique cohesinindependent cohesion mechanism to hold sister chromatids together before separation, and behaves in unique ways with respect to replication, recombination and transcription. These rDNAspecific features may promote a chromosome segregation mechanism distinct from the rest of the genome. Therefore, the aim of this thesis was to test the hypothesis that the rDNA affects chromosome segregation. To test this hypothesis I focused on mitotic chromosome segregation, and used the model genetic organism, Saccharomyces cerevisiae. S. cerevisiae offers many advantages for testing this hypothesis, including its tolerance to aneuploidy and systems that have been developed to genetically manipulate the rDNA. I developed and optimized a chromosome loss assay (CLA) that measures the rate of chromosome loss during mitosis in S. cerevisiae. I modified a number of strains that had alterations associated with the rDNA, including strains deleted for the chromosomal rDNA repeats, with a reduction in rDNA copies, and with the rDNA translocated to a different chromosome, with specific phenotypic markers for detection of chromosome loss events. I then tested the chromosome loss rates of these strains using the CLA. My results demonstrate that the rDNA affects mitotic chromosome segregation fidelity at two levels. First, the rDNA increases the segregation fidelity of the rDNA-containing chromosome, defining a local chromosome segregation role for the rDNA. I found that this local effect is mediated by the rDNA binding protein Fob1, and I propose three potential mechanisms for how Fob1 mediates this role: (1) through establishment of rDNA recombination-intermediates that may help to stabilize the long rDNA locus; (2) through recruitment of condensin to establish intra-chromatid linkages that promote timely condensation of sister chromatids; or (3) through recruitment of a silencing complex to achieve an appropriate rDNA chromatin state for chromosome segregation. Second, I show that the rDNA has a global effect on chromosome segregation fidelity, with rDNA deletion or reduction in rDNA copies influencing the segregation of many or all chromosomes. Curiously, heterozygosity of rDNA state, regardless of what states are present, confers wild type missegregation rates. I rule out trivial explanations for this global effect, and instead propose that the rDNA affects segregation through changes in nucleolar structure and overall nuclear organization that impact spindle polarity and thus the fidelity of chromosome segregation. Together, these results define a new role for the rDNA in facilitating chromosome segregation, and one that acts at two different levels. This work provides insights into a novel beneficial role of the rDNA in chromosome segregation of S. cerevisiae, and the conserved mechanism of chromosome segregation across eukaryotes suggests the rDNA may play similar roles in more complex organisms. It will be interesting to determine if the rDNA also has beneficial role in meiosis, where the rDNA has been associated with missegregation.