The Tof1p–Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae

Abstract

Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase (''sweepase''). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p. Consistent with this mechanism, very few replication forks were arrested at the natural replication termini in the absence of the two checkpoint proteins. In the absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes restored fork arrest by removing both the fork-releasing and fork-protection activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in the MRC1 checkpoint pathway were not involved in this global control. This observation suggests that Tof1p-Csm3p function differently from MRC1 and the other above-mentioned genes. This mechanism is not restricted to the natural Ter sites but was also observed at fork arrest caused by the meeting of a replication fork with transcription approaching from the opposite direction.protein-protein interaction ͉ replication terminus ͉ terminator protein S ite-specific replication termini (Ter sites), also called replication fork barriers, are present in many prokaryotic chromosomes and at certain specific regions of eukaryotic chromosomes, such as the nontranscribed spacers of rDNA of Saccharomyces cerevisiae (Fig. 1A;. Binding of the cognate replication terminator proteins to the Ter sites causes programmed fork arrest that has special physiological functions (4). The terminator proteins of prokaryotes antagonize the activity of the replicative hexameric helicases in a polar mode (5-7), not only by binding to the Ter DNA but also by protein-protein contact with the replicative helicase (8, 9). Several aspects of the mechanism of fork arrest have been reviewed (4, 10).Eukaryotic replication termini are located in the nontranscribed spacers of rDNA of both budding and fission yeast (2,3,11,12). A protein called Fob1p is necessary for fork arrest at the tandem Ter sites present at the nontranscribed spacers of S. cerevisiae (13,14). A number of Ter-binding proteins have been discovered in Schizosaccharomyces pombe to date that bind to the replication termini located near the mating type locus and at the nontranscribed spacers of rDNA (15)(16)(17)(18)(19). Whether these proteins, like their analogues in prokaryotes, terminate replication by antagonizing the replicative helicase is not known at this time.In addition to the terminus-binding protein, two checkpoint proteins ca...

Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

“…Since deletion of ScPIF1 leads to an increase in mitochondrial DNA point mutations, particularly after oxidative damage (21,51,52,67), it is also possible mPif1 plays a nonessential role in mitochondrial genome stability. Although Scrrm3⌬ cells do not exhibit changes in GCR or mitochondrial genome stability, these cells do exhibit genetic instability, presumably due to replication fork pausing at specific chromosomal loci, such as the ribosomal DNA locus (31,32,45,59,68,69). ScRRM3 and ScPIF1 exhibit genetic interactions (including synthetic lethality) with many gene products required in DNA replication and S-phase checkpoint arrest (1,2,17,50,58,59,69,71).…”

“…Unlike Rrm3 and Pif1, Pfh1 is essential (77). Rrm3 promotes the progression of replication forks at particular chromosomal loci prone to pausing during DNA replication (including the telomere), and rrm3⌬ cells exhibit a modest telomere lengthening (11,32,33,45,59,69). Mutation of ScPif1 leads to telomerase-dependent telomere lengthening (33,76), and this phenotype depends on the ATPunwinding activity of ScPif1; the enzyme has a preference for short (Ͻ30 nucleotides) RNA-DNA hybrids and can unwind telomerase RNA from a telomeric DNA substrate (15).…”

Transcription regulatory elements are punctuation marks for DNA replication

“…Clearly, however, the head-on collisions with either elongating or initiating RNA polymerase have much more dire consequences for DNA replication. It is tempting to speculate, therefore, that the front edge of the RNA polymerase could be a potent contrahelicase (45).…”

Tipin and Timeless form a mutually protective complex required for genotoxic stress resistance and checkpoint function

“…Like their mammalian counterpart, Csm3 and Swi3 have been shown to interact with Tof1 and Swi1, respectively, and are also required for activation of Rad53 and Cds1 in response to HU (19)(20)(21)(22)(23)(24). Like Tof1, Csm3 was found to be required for the pausing of replication forks at replication fork barriers and site-specific replication termini in yeast (15,25). Recently, human Timeless has been reported to play a role in the DNA damage checkpoint response (26).…”