The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae - PubMed (original) (raw)

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

Bidyut K Mohanty et al. Proc Natl Acad Sci U S A. 2006.

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.

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Figures

Fig. 1.

Diagrammatic representation of a single repeat of the rDNA array of S. cerevisiae and a model of a terminated replication fork. (A) The repeated sequence present in the rDNA showing the 35S region transcribed by RNA polymerase I, the 5S region transcribed by RNA polymerase III, nontranscribed spacer I (NTS1) that contains tandem Ter sites and the nontranscribed spacer II (NTS2) that contains an origin of bidirectional replication (ARS). (B) A model of a terminated fork showing the locations of the two Ter sites that are bound to Fob1p, the putative replicative helicase MCM2–7p, the proliferating cell nuclear antigen sliding clamp (lavender ring), the Tof1p–Csm3p protein complex that either directly or indirectly antagonizes the Rrm3p helicase that progresses 5′ to 3′ on DNA and tends to release the terminated forks probably by transiently displacing Fob1p.

Fig. 2.

Autoradiograms of 2D gels showing the role of Tof1p on fork arrest at Ter1 and -2. The Ter spots in WT yeast (arrow) are almost completely abolished in Δ_tof1_ cells. There was failure to complement (and thereby restore the Ter spot) by a plasmid expressing Fob1p in Δ_tof1_ cells and complementation of Δ_tof1_ cells by plasmid-borne WT TOF1.

Fig. 3.

Autoradiogram of 2D gels of replication intermediates of rDNA in Δ_mrc1_, Δ_wss1_, Δ_psy2_ strains showing that deletions of these genes had no effect on replication termination (all of the samples showed distinct termination spots).

Fig. 4.

Autoradiograms of 2D gels showing termination activity in WT strain and its derivatives. (A) The Ter spot is slightly enhanced (≈2-fold) in Δ_rrm3_ cells in comparison with the WT but is mostly missing in Δ_tof1_ and Δ_csm3_ cells. The Δ_tof1_Δ_rrm3_ and Δ_csm3_Δ_rrm3_ double deletions at least partially restored the Ter spot. Black arrowheads show the Ter spot(s). The unfilled arrowhead indicates fork stalling on the descending part of the arc in Δ_rrm3_ cells. (B) In Δ_sgs1_ cells, a ≈2-fold enhancement of Ter spot over that of the WT was observed. Simultaneous deletions of both sgs_1 and tof1 (Δ_sgs1_Δ_tof1) caused disappearance of the Ter spot; removal of srs2 (Δ_srs2_) did not reduce fork arrest at Ter; double deletions, Δ_srs2_ Δ_tof1_ did not cause restoration of fork arrest not unlike in the Δ_sgs1_Δ_tof1_ cells; Δ_srs2_Δ_rrm3_ Δ_rad51_ (Δ_rad51_ was included to overcome synthetic lethality of Δ_srs2_Δ_rrm3_) did not affect the frequency of fork arrest, whereas the quadruple deletion that also contained Δ_tof1_ shows reduced fork arrest.

Fig. 5.

ChIP analysis of fork arrest at the Ter sites in different genotypes. (A) Diagram showing the location of the primer pairs used to amplify Ter-specific and control DNA fragments that were crosslinked to myc-tagged minichromosome maintenance protein (MCM7p) and immunoprecipitated by anti-myc antibodies. (B) A representative photograph of the of the PCR products resolved in a 6% polyacrylamide gel. (C) Quantification of the ratios of the (from three independent sets of experiments) Ter fragment over that of the control fragment. Intensities of the DNA bands were calculated by using an image scanner and the

imagequant

software of ethidium bromide-stained gels. Computing the ratio of intensity of Ter band over that of the control band and normalizing that by multiplying with the inverse of the corresponding ratio of the input DNA gave the relative enrichment index. The primer pairs used are shown in Supporting Text.

Fig. 6.

Effects of Tof1p and Rrm3p on forks stalled by encounter between a RNA polymerase III transcription complex and a fork coming from the opposite direction. (Top) Diagram showing the structure of the plasmid pAT15B. (Middle and Bottom) Autoradiograms of 2D gels showing that encounter between transcription emanating from a tRNA promoter (by RNA pol III) and the fork moving counterclockwise from the ARS generate two elongated spots in WT cells (see parentheses and arrow); the spots are either missing or greatly attenuated in the Δ_tof1_ and significantly enhanced in the Δ_rrm3_ cells. Double deletion of the two genes (see Δ_rrm3_Δ_tof1_) restored the forks to a level significantly higher than that of the WT cells.

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The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae - PubMed (original) (raw)