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
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Date
2016
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Massey University
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.
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Keywords
Saccharomyces cerevisiae, Genetics, Recombinant DNA, Chromosome abnormalities, Mitosis, Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Genetics