SOMATIC LOSS OF MATERNAL POLA2 CAUSES TISSUE-DEPENDENT CELL DEATH AND DNA DAMAGE IN POLA2 MUTANT ZEBRAFISH
Open Access
- Author:
- Lin, Alex Yu
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 16, 2018
- Committee Members:
- Keith C Cheng, Dissertation Advisor/Co-Advisor
Keith C Cheng, Committee Chair/Co-Chair
Kristin Ann Eckert, Committee Member
Douglas B Stairs, Committee Member
Ralph Lauren Keil, Outside Member - Keywords:
- DNA polymerase alpha
huli hutu
hht
zebrafish
DNA damage
Cell cycle arrest
Cell death - Abstract:
- DNA replication is essential to all life. Loss of function mutations of POLA2, a critical component of DNA replication, in Saccharomyces cerevisiae and Arabidopsis thaliana causes rapid growth arrest. Due to the unicellular nature of S. cerevisiae and embryonic lethality in A. thaliana beyond the 2-cell stage, cell type-dependent effects of somatic deficiency of POLA2 on cell morphology and cellular functions have not been well elucidated. A zebrafish genetic screen for mutations that cause nuclear atypia yielded a mutant, huli hutu (hht), with a pleiotropic phenotype that included nuclear atypia in gastrointestinal cells, apoptotic nuclear fragmentation in the neurons of the brain and eyes, reduced head and eye size, and dorsal curvature of the body. The causative frameshift mutation in pola2, which encodes subunit B of DNA polymerase α (Pol α), results in a premature stop codon at the 38th amino acid position of the 600-amino acid protein. Cell cycle and DNA synthesis analyses indicated that loss of pola2 significantly increased the proportion of cells in S-phase and reduced DNA synthesis in hht larvae. DNA damage and cell death were localized to neuronal cells of the brain, eyes, and spinal cord of the hht mutants. The observation of these phenotypes were possible due to the extended 5-7 day survival of the hht fish, which stands in striking contrast with the lethality of the corresponding mutants in yeast and Arabidopsis. Our data suggest this difference is likely explained by the presence of wild-type maternal pola2 in homozygous mutant embryos that supports active DNA synthesis during early embryogenesis. The subsequent depletion of wild-type pola2 leads to defective DNA synthesis, DNA damage, cell death, and cell cycle arrest that result in the tissue-dependent cytological deformities observed in hht.