Regulation of DNA Replication During Conventional and Unconventional Cell Cycles in Tetrahymena

Abstract

As the nucleating protein for pre-replicative complex (pre-RC) assembly, the conserved Origin Recognition Complex (ORC) specifies where replication initiates in eukaryotic chromosomes. During the vegetative cell cycle of Tetrahymena thermophila, previously published work has shown that DNA replication initiates from defined chromosomal sites in an ORC-dependent manner. Tetrahymena exhibits nuclear dimorphism, a polyploid somatic macronucleus (MAC), which is transcriptionally active and maintains vegetative growth, and a diploid germline micronucleus (MIC) responsible for the transmission of genetic information during conjugation. In order to provide more information about the fundamental mechanisms of micro- and macro- nuclear replication programs, I study the impacts of changing in ORC protein contents on the fate of micro- and macro- nuclear chromosomes during the vegetative cell cycle and development in Tetrahymena.

I examined the effect of down-regulation of ORC1 on genome stability and intra- S phase checkpoint activation by disrupting ORC1 gene in the macronucleus. Partial depletion of Orc1p leads to genome instability in the diploid mitotic micronucleus, abnormal division of the polyploid amitotic macronucleus, and failure to mount a robust intra-S phase checkpoint response. In addition, the ORC1 knockdown strain fails to execute two developmentally- regulated DNA replication programs, endoreplication and ribosomal DNA (rDNA) gene amplification. I also examined the regulation of ORC and MCM during development. Remarkably, the result suggests that the demand on the ORC-dependent replication machinery differs during development and the vegetative S phase.

To further gain new insights into fundamental mechanisms that protect chromosomes from replication stress, I examined the impact of replication stress on the regulation of ORC and MCM. This study led to the discovery of a novel DNA replication program that is activated under HU treatment. While Orc1p and Mcm6p were selectively degraded in response to HU, cells were competent to complete S phase in the absence of Orc1p and Mcm6p after HU was removed. In addition, the rDNA origin used exclusively during the S phase of vegetative cell cycle and developmentally programmed gene amplification is suppressed when these replication proteins are selectively degraded under HU treatment. Instead, an alternative program was used to resume the cell cycle progression. These data provide compelling evidence for an ORC-independent DNA replication program in cells recovering from replication stress.