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

摘要

The Tetrahymena thermophila rDNA minichromosome has been used as a modelsystem for studying DNA replication. Previous studies have identified cis-actingreplication determinants within the rDNA origin and promoter region including the type Ielement 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 playa role in selectively marking these regions. In this dissertation, I use gene disruption toelucidate the role of TIF1 in replication. This work reveals that TIF1 represses rDNAorigin firing, and is required for proper macronuclear S phase progression and division.Replication at the rDNA origin initiates precociously despite the observation that TIF1mutants exhibit an elongated macronuclear S phase and a diminished rate of DNAreplication. The amitotic macronucleus also displays delayed and abnormal division eventhough cells exit S phase with a wild-type macronuclear DNA content. Nuclear defects arealso evident in the diploid micronucleus as TIF1 mutants contain fewer micronuclearchromosomes and are unable to pass genetic information to progeny. This defect isprogressive as clonal mutant lines exhibit micronuclear instability during subsequentvegetative 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 damagingagents. This suggests that TIF1 mutants may have defects in the DNA damage responsepathway. TIF1-deficient cells also incur DNA damage with no exogenous damagingagents. I propose that micro- and macronuclear defects witnessed in TIF1 mutant cellsresult from cells exiting S phase with compromised chromosomes due to the accumulationof DNA damage. Furthermore, TIF1 appears to play a role in the prevention, recognitionor repair of DNA damage in addition to regulating rDNA replication and cell cycleprogression and division. Additionally, TIF1 plays an essential role in the faithfulpropagation of both the macro- and micronuclear genomes.