Distinct DNA Replication Programs in Tetrahymena Thermophila
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DNA replication is a vital process to duplicate genetic material for inheritance. A conventional mitotic cell cycle is composed of G1, S, G2 and M phases, and DNA is replicated during S phase. Besides the conventional cell cycle, there are other distinct replication programs. Here in my dissertation, I used the organism Tetrahymena thermophila to study DNA replication because it employs alternative DNA replication programs, such as genome-wide endoreplication, locus-specific gene amplification and an unprecedented DNA replication program in cells that are recovered from hydroxyurea (HU) induced replication stress. In my dissertation research, I determined that ribosomal DNA (rDNA) minichromosome amplification occurs when non-rDNA chromosomes are undergoing endoreplication during Tetrahymena development, and that both programs are shut down simultaneously. I found that rDNA amplification is then switched to endoreplication upon refeeding when the levels of the initiation proteins, the origin recognition complex (ORC) and the minichromosome maintenance protein complex (MCM2-7) are dramatically reduced. During this stage, the rDNA origin is not utilized, and a higher origin density was observed on a genome-wide scale. These data indicate that origin utilization is altered for endoreplication and suggest an ORC-independent initiation mechanism. More importantly, rDNA replication intermediates that are accumulated in both wild type endoreplication and vegetative S phase of a histone monomethyltransferase defective strain TXR1Δ share the same signature, suggesting epigenetic modifications may be involved in replication initiation and elongation during endoreplication. As part of my dissertation research, I studied a DNA replication program that occurs after ORC and MCM proteins are degraded in hydroxyurea treated cells. I found that replication forks are arrested rather than slowed down upon HU treatment, when the protein levels of ORC and MCMs are degraded. I detected new origin firing on a genome-wide scale upon HU removal, before the protein levels of ORC and MCMs are restored. Moreover, the rDNA origin that is used for vegetative S phase is not utilized in this specialized replication program. The collective data suggest that an ORC-independent initiation is utilized. In summary, my research has led to new discoveries of distinct DNA replication initiation and elongation mechanisms in eukaryotes.
Meng, Xiangzhou (2015). Distinct DNA Replication Programs in Tetrahymena Thermophila. Doctoral dissertation, Texas A & M University. Available electronically from