Roles of the Tetrahymena thermophila type I element binding factor, TIF1, in DNA replication and genome stability

The Tetrahymena thermophila rDNA minichromosome has been used as a model system for studying DNA replication. Previous studies have identified cis-acting replication determinants within the rDNA origin and promoter region including the type I element that is essential for replication initiation, fork progression and promoter activation. TIF1 is a non-ORC single strand-binding protein that binds the type I element in vivo. TIF1 binds opposing strands at the origin and promoter regions indicating that it may play a role in selectively marking these regions. In this dissertation, I use gene disruption to elucidate the role of TIF1 in replication. This work reveals that TIF1 represses rDNA origin firing, and is required for proper macronuclear S phase progression and division. Replication at the rDNA origin initiates precociously despite the observation that TIF1 mutants exhibit an elongated macronuclear S phase and a diminished rate of DNA replication. The amitotic macronucleus also displays delayed and abnormal division even though cells exit S phase with a wild-type macronuclear DNA content. Nuclear defects are also evident in the diploid micronucleus as TIF1 mutants contain fewer micronuclear chromosomes and are unable to pass genetic information to progeny. This defect is progressive as clonal mutant lines exhibit micronuclear instability during subsequent vegetative cell cycling. This work reveals that these macro- and micronuclear phenotypes may be the result of DNA damage as TIF1 mutants are hypersensitive to DNA damaging agents. This suggests that TIF1 mutants may have defects in the DNA damage response pathway. TIF1-deficient cells also incur DNA damage with no exogenous damaging agents. I propose that micro- and macronuclear defects witnessed in TIF1 mutant cells result from cells exiting S phase with compromised chromosomes due to the accumulation of DNA damage. Furthermore, TIF1 appears to play a role in the prevention, recognition or repair of DNA damage in addition to regulating rDNA replication and cell cycle progression and division. Additionally, TIF1 plays an essential role in the faithful propagation of both the macro- and micronuclear genomes.