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Open Phd Dissertation - Alex Yu The Pennsylvania State University The Graduate School College of Medicine SOMATIC LOSS OF MATERNAL POLA2 CAUSES TISSUE-DEPENDENT CELL DEATH AND DNA DAMAGE IN POLA2 MUTANT ZEBRAFISH A Dissertation in Cell and Molecular Biology by Alex Y. Lin Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2019 The dissertation of Alex Y. Lin was reviewed and approved* by the following: Keith C. Cheng Distinguished Professor of Pathology, Biochemistry and Molecular Biology, and Pharmacology Chief, Division of Experimental Pathology Dissertation Advisor Chair of Committee Kristin A. Eckert Professor of Pathology, and Biochemistry and Molecular Biology Ralph L. Keil Chair, Biomedical Sciences Graduate Program Associate Professor of Biochemistry and Molecular Biology Douglas B. Stairs Assistant Professor of Pathology and Pharmacology Charles Lang Associate Dean, Graduate Studies Distinguished Professor of Cellular and Molecular Physiology and Surgery *Signatures are on file in the Graduate School ii 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. iii Table of Contents Page List of Figures…………………………………………………………………………....vii List of Tables…………………………………………………………………………....viii List of Abbreviations…………………………………………………………………......ix Acknowledgements………………………………………………………………………..x Chapter 1 ............................................................................................................................. 1 1.1 Nuclear atypia in cancer ............................................................................................ 1 1.2 Zebrafish forward genetic screen .............................................................................. 7 1.3 Identification of the hht mutant ............................................................................... 13 1.4 Identification of the causative mutation in hht ........................................................ 16 1.5 DNA polymerase α-primase complex and POLA2 ................................................. 20 1.5.1 Pol α and related polymerases .......................................................................... 20 1.5.2 Primary function of Pol α ................................................................................. 20 1.5.3 Pol α structure ................................................................................................... 21 1.5.4 Other functions of Pol α subunits ..................................................................... 26 1.5.5 Functions of the B subunit ................................................................................ 30 1.6 Statement of thesis .................................................................................................. 32 Chapter 2 ........................................................................................................................... 34 2.1 Fish lines, mating, and embryo collection............................................................... 34 2.2 Processing for histology .......................................................................................... 34 2.3 Sequencing .............................................................................................................. 35 2.4 Cell cycle analysis ................................................................................................... 35 2.5 Tol2-mediated transgenesis ..................................................................................... 35 2.6 Putative pola2 promoter .......................................................................................... 37 2.7 Chemical inhibition of replication........................................................................... 37 2.8 Acridine orange staining ......................................................................................... 38 2.9 γ-H2AX staining ..................................................................................................... 38 2.10 EdU staining .......................................................................................................... 39 2.11 Detection of wild-type pola2 transcript ................................................................ 39 2.12 Bioinformatics ....................................................................................................... 39 2.12.1 Sequence homology analysis .......................................................................... 39 iv 2.12.2 Syntenic analysis ............................................................................................ 40 2.12.3 Conserved domains analysis ........................................................................... 40 2.13 DNA polymerase mutant generation using CRISPR/Cas9 ................................... 40 2.14 Statistics ................................................................................................................ 41 Chapter 3 ........................................................................................................................... 42 3.1 Introduction ............................................................................................................. 42 3.2 Validation of the causative mutation of hht ............................................................ 43 3.3 pola2-deficiency causes accumulation of cells in S phase ...................................... 55 3.4 pola2-deficiency induces localized cell death ......................................................... 57 3.5 pola2-deficiency causes localized DNA damage .................................................... 62 3.6 pola2-deficient mutants exhibit reduced DNA synthesis ........................................ 67 3.7 Wild-type maternal pola2 mRNA sustains survival of hht ..................................... 72 3.8 Discussion ............................................................................................................... 75 3.8.1 Extended survival of hht is supported by wild-type pola2 mRNA ................... 75 3.8.2 Source of the wild-type pola2 mRNA .............................................................. 75 3.8.3 Is wild-type pola2 mRNA critical to the extended survival of hht? ................. 76 3.8.4 Presence of wild-type Pola2 protein in hht embryos ........................................ 77 3.8.5 Pola2-deficiency and cell death ........................................................................ 78 3.8.6 Timing of Cell death, DNA damage (double-stranded breaks), and reduced DNA synthesis ........................................................................................................... 79 3.8.7 Model: sequence of events that lead to the hht phenotype ............................... 80 Chapter 4 ........................................................................................................................... 81 4.1 Summary ................................................................................................................. 81 4.2 DNA synthesis-deficiency in hht ............................................................................ 82 4.3 Tissue-dependent phenotype in hht ......................................................................... 85 4.3.1 Cell proliferation and tissue-dependent phenotypes ......................................... 86 4.3.2 Tissue-dependent DNA repair .......................................................................... 89 4.4 Pola2-deficiency and DNA damage ........................................................................ 90 4.4.1 Inhibition of DNA replication and double-stranded breaks ............................. 90 4.4.2 How does reduced DNA replication lead to double-stranded break? .............. 91 4.4.3 Generation of single-stranded
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