ABSTRACT
The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus.
Subject(s)
Diploidy , Macronucleus/metabolism , Micronucleus, Germline/metabolism , Nuclear Proteins/metabolism , Polyploidy , S Phase , Tetrahymena/cytology , Transcription Factors/metabolism , Animals , Caffeine/pharmacology , Chromosomes/drug effects , Chromosomes/metabolism , DNA Damage , Gene Expression Regulation/drug effects , Genomic Instability/drug effects , Macronucleus/drug effects , Meiosis/drug effects , Methyl Methanesulfonate/toxicity , Micronucleus, Germline/drug effects , Mitosis/drug effects , Mutation/genetics , Neomycin , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Protein Transport/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rad51 Recombinase/metabolism , S Phase/drug effects , Tetrahymena/drug effects , Transcription Factors/deficiency , Transcription Factors/geneticsABSTRACT
The non-ORC protein, TIF1, recognizes sequences in the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome that are required for origin activation. We show here that TIF1 represses rDNA origin firing, but is required for proper macronuclear S phase progression and division. TIF1 mutants exhibit an elongated macronuclear S phase and diminished rate of DNA replication. Despite this, replication of the rDNA minichromosome initiates precociously. Because rDNA copy number is unaffected in the polyploid macronucleus, mechanisms that prevent reinitiation appear intact. Although mutants exit macronuclear S with a wild-type DNA content, division of the amitotic macronucleus is both delayed and abnormal. Nuclear defects are also observed in the diploid mitotic micronucleus, as TIF1 mutants lose a significant fraction of their micronuclear DNA. Hence, TIF1 is required for the propagation and subsequent transmission of germline chromosomes. The broad phenotypes associated with a TIF1-deficiency suggest that this origin binding protein is required globally for the proper execution and/or monitoring of key chromosomal events during S phase and possibly at later stages of the cell cycle. We propose that micro- and macronuclear defects result from exiting the respective nuclear S phases with physically compromised chromosomes.
