TIF1 Represses rDNA Replication Initiation, but Promotes Normal S Phase Progression and Chromosome Transmission inTetrahymena

Abstract

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. INTRODUCTIONThe initiation of eukaryotic DNA replication is regulated by protein-DNA interactions that occur within defined chromosomal domains, termed replicators or replicons. Eukaryotic replicators are modular and contain binding sites for the conserved six-subunit origin recognition complex (ORC;Bell and Stillman, 1992) and non-ORC DNA binding proteins (Marahrens and Stillman, 1992). ORC plays a central role, recruiting proteins involved in replication initiation and elongation to form the prereplicative complex (pre-RC). These proteins include a replicative helicase-the minichromosome maintenance (MCM) complex (Ishimi, 1997;Labib et al., 2000) and factors that regulate origin activation, such as Cdc6 and Cdt1 (Nishitani et al., 2000;Oehlmann et al., 2004).Although ORC binding sites are plentiful in the Saccharomyces cerevisiae (Sc) genome, only a fraction are routinely engaged in replication initiation (Theis and Newlon, 1993;Wyrick et al., 2001). Genetic studies indicate that ScORC binding is necessary, but not sufficient, to confer replicator status to a chromosomal domain. Although ScORC binds DNA in a sequence-specific manner, metazoan ORCs exhibit no obvious sequence-specificity, displaying a preference for degenerate, asymmetric A:T-rich sequences in vitro (Austin et al., 1999;Chesnokov et al., 2001;Vashee et al., 2001) and in vivo (Kong et al., 2003;Vashee et al., 2003). This relaxed specificity is similar to Schizosaccharomyces pombe (Sp) ORC, which binds DNA via an unusual A-T hook domain in its Orc4 subunit (Chuang and Kelly, 1999;Kong and DePamphilis, 2001). Although metazoan ORCs lack AT hooks, they still associate with specific replicator domains in vivo (Austin et al., 1999;Abdur...

TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus ofTetrahymena

“…Most significantly, we demonstrate that Tif1p is required to activate the intra-S-phase checkpoint response in both nuclear compartments. These observations, in conjunction with the previously described role for Tif1p at the rDNA origin (Morrison et al, 2005), suggest that this protein contributes to chromosome homeostasis through its action at origins and at elongating replication forks.…”

“…We obtained preliminary evidence for a macronuclear S-phase checkpoint during our analysis of tif1-1::neo mutants, which among other things are partially defective in macronuclear S-phase progression (Morrison et al, 2005). TIF1-deficient cells exhibit a prolonged macronuclear S phase, that once complete, is followed by a further delay in macronuclear division and cytokinesis (Morrison et al, 2005).…”

“…TIF1-deficient cells exhibit a prolonged macronuclear S phase, that once complete, is followed by a further delay in macronuclear division and cytokinesis (Morrison et al, 2005). Whereas macronuclear divisions are frequently aberrant in TIF1 mutants, the observed defect in S phase progression and subsequent delay in macronuclear division and cytokinesis argue that macronuclear and cytoplasmic cell cycles are coordinately regulated.…”

“…Whereas macronuclear divisions are frequently aberrant in TIF1 mutants, the observed defect in S phase progression and subsequent delay in macronuclear division and cytokinesis argue that macronuclear and cytoplasmic cell cycles are coordinately regulated. Tif1p binds essential cisacting replication determinants in the rDNA origin, in vitro and in vivo Saha et al, 2001) and was recently shown to regulate rDNA origin activation, functioning in a repressive capacity to prevent precocious initiation during macronuclear S phase (Morrison et al, 2005). Because macronuclear S phase is prolonged in Tif1p-deficient strains, Tif1p might regulate initiation and/or the elongation of replication forks in other (non-rDNA) macronuclear chromosomes.…”

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

“…1B) Flow cytometry. Tetrahymena cells were lysed in a buffer containing 0.25 M sucrose, 10 mM MgCl 2 , and 0.5% Triton X-100 at a concentration of 1 ϫ 10 6 cells/ml (35). Propidium iodide was added to 10 g/ml, and nuclei were stained for 30 min prior to analysis using a FACSCalibur instrument.…”