טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentWeindling Esther
SubjectRole and Regulation of Candida Albicans NRM1 in Cell Cycle
Progression
DepartmentDepartment of Medicine
Supervisor Professor Daniel Kornitzer
Full Thesis textFull thesis text - English Version


Abstract

Candida albicans is a commensal microorganism that can cause deep-seated, life-threatening infection among immunocompromised or debilitated patients. CaNrm1 is the C. albicans homolog of the S. cerevisiae Whi5 and Nrm1 transcription inhibitors that, analogous to mammalian Retinoblastoma, regulate the cell cycle transcription program in the G1 phase and at the G1/S transition. Deletion of CaNRM1 has been previously shown to cause a reduction in cell size (similar to the whi5 phenotype), an increase in resistance to the DNA replication inhibitor hydroxyurea (HU) (similar to the nrm1 phenotype), and derepression of G1 and S phase-specific genes. Previous genetic epistasis analysis had suggested that CaNrm1 interacts with the C. albicans MBF transcription factor (CaSwi4/CaSwi6). The transcriptional response to HU had been shown to require CaNrm1, but the regulatory mechanism was unknown.

This work addressed the post-transcriptional regulation of CaNrm1, namely at the level of protein stability, subcellular localization, and protein modification. We showed that CaNrm1 is normally unstable and that its degradation is inhibited under DNA replication stress condition (addition of HU), which arrests the cells in S phase. Degradation occurs very rapidly in cells arrested in G2/M by the antimitotic drug nocodazole, and could not be rescued by the addition of HU, suggesting that effects of both of these drugs on CaNrm1 are a consequence of their effect on cell cycle position of the cell population. Concerning subcellular localization of CaNrm1, we found that it is excluded from the nucleus in G1 (unbudded) cells. The use of S-phase inhibitors (HU and methylmethane sulfonate) led to a stronger nuclear localization.

We found that CaNrm1 interacts physically with the MBF via CaSwi4. The apparent paradox of higher stability and more pronounced nuclear localization of CaNrm1 under replication stress, conditions that should inactivate CaNrm1 and derepress MBF activity, was solved by our finding that the physical interaction between CaNrm1 and CaSwi4 was disrupted under these conditions, possibly by phosphorylation of CaSwi4. In an attempt to identify kinases that modify CaNrm1 itself we isolated CaYck2. CaYck2 overexpression affects CaNrm1 modification and nuclear localization, however its role in CaNrm1 regulation was not elucidated.