UBC Theses and Dissertations
Identification of novel factors required for chromosome segregation in budding yeast Cheng, Benjamin
During the course of the mitotic cell cycle, the genetic material must be faithfully replicated and segregated to daughter cells. After DNA replication when chromosomes have been duplicated, each pair of identical sister chromatids must remain tethered together until all pairs of sister centromeres have attached to the mitotic spindle in a bi - oriented manner, a state termed metaphase. Once metaphase has been successfully achieved, the initiation of anaphase can take place, and sister chromatids are pulled apart to the two daughter cells. Errors in this process lead to chromosome missegregation (chromosome loss or non-disjunction) and result in aneuploidy, which may have deleterious effects. Processes important in chromosome segregation fidelity include kinetochore attachment to the spindle and sister chromatid cohesion. A genome wide two hybrid screen using SGT1 as the "bait" identified a previously uncharacterized open reading frame, YDR014W (RAD61) that, when deleted, missegregated a chromosome fragment. YDR014W corresponded to the gene encoding the complementation group, CTF6, and was also recently characterized as RAD61 in a screen for deletion mutants sensitive to ionizing radiation. rad61Δ diploid mutant strains displayed a G2/M progression delay dependent on Mad2p and were hypersensitive to DNA damaging agents. Rad61p localizes to the nucleus and a fraction binds chromatin. Rad61p is not a core component of the yeast kinetochore and is not required for homologous recombination repair of DNA damage, but is important for sister chromatid cohesion. Using co-immunoprecipitation and mass spectrometry analysis, we identified a protein-protein interaction between Rad61p and Dedlp, an RNA helicase of the DEAD box family that has important roles in initiation of translation and mRNA splicing. Dedlp binds chromatin and may have direct roles in chromosome biology. A temperature sensitive allele of the essential DED1 gene causes an increased rate of chromosome missegregation. rad61Δ and dedl temperature sensitive alleles displayed conditional synthetic lethality, indicating that the interaction is functionally significant within yeast cells. Taken together, these results suggests that Rad61p and Dedlp function together in the nucleus for processes that are important for sister chromatid cohesion and for chromosome segregation.
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