Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of Saccharomyces cerevisiae

Abstract

The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable. First, a Fob1 ortholog of Saccharomyces bayanus expressed in a fob1Delta strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of S. cerevisiae Fob1 retained normal fork arresting activity but was partially defective in rDNA silencing. We further show that the silencing defect of S. bayanus Fob1 and the Iota407Tau mutant of S. cerevisiae Fob1 were caused by the failure of the proteins to interact with two members of the S. cerevisiae RENT complex, namely S. cerevisiae Sir2 and S. cerevisiae Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.

FIGURE 1.

Model showing termination and silencing functions of Fob1 in rDNA of S. cerevisiae. A, rDNA array in chromosome XII, each rDNA unit has 35 S and 5 S-encoding sequences punctuated by two nontranscribed spacers. Note the ARS in spacer 2 and twin Ter sites in spacer 1. B, shown is the protein complex containing Fob1, Tof2, Csm1, Lrs4, and the RENT complex (containing Net1, Cdc14, and Sir2). C, shown is a schematic representation of Fob1-mediated fork arrest at the Ter sites. In C, the protein components necessary for fork arrest are shown, and the silencing complex has been omitted to simplify the picture. It does not imply that the silencing complex has to be removed before fork arrest occurs; D, shown is the rDNA-silencing RENT complex consisting of the indicated component proteins. ars, origin of replication; Pr, promoter.

FIGURE 2.

Separation of replication fork arrest and silencing functions of Fob1 in S. cerevisiae orthologs. A, two-dimensional agarose gel analysis of replication intermediates from a fob1Δ derivative of S. cerevisiae (YSB348) strain containing vector (pGAD424) alone, S. cerevisiae Fob1 (Sc Fob1), S. bayanus Fob1 (Sb Fob1), and S. paradoxus Fob1 (Sp Fob1). B, schematic diagram showing the silencing cassette (mURA3) located 50-bp downstream of the single Ter2 site present at the end of rDNA array in chromosome XII. C, silencing assay in fob1ΔYSB348 containing Fob1 orthologs. The data show that S. cerevisiae Fob1 and S. paradoxus Fob1 are proficient in silencing, but S. bayanus Fob1 is defective in silencing of the expression of the mURA3 cassette.

FIGURE 3.

Two-hybrid interactions of Fob1 orthologs with S. cerevisiae Net1 and S. cerevisiae Sir2 showing defect in the protein-protein interaction of S. bayanus Fob1. A, interaction of pGAD424 S. cerevisiae Fob1 (ScFob1) with pGBT9 S. cerevisiae Net1 (ScNet1) and pGBT9 S. cerevisiae Sir2 (ScSir2). B, interaction of pGAD424 S. paradoxus Fob1 (SpFob1) with pGBT9 S. cerevisiae Net1 (ScNet1) and pGBT9 S. cerevisiae Sir2. C, interaction of pGAD424 S. bayanus Fob1 (SbFob1) with pGBT9 S. cerevisiae Net1 and pGBT9 S. cerevisiae Sir2. D and E, β-galactosidase assay of yeast two-hybrid interactions of S. cerevisiae Fob1 and S. bayanus Fob1 with S. cerevisiae Net1 and S. cerevisiae Sir2, respectively.

FIGURE 4.

Separation of fork arresting and silencing activities in the S. cerevisiae Fob1 I407T mutant. A, silencing of mURA3 in the fob1Δ strain containing vector (pGAD424), S. cerevisiae Fob1 (ScFob1), and its I407T derivative. B and C, two-hybrid interactions of S. cerevisiae Fob1 or its I407T derivative with S. cerevisiae Net1 and S. cerevisiae Sir2, respectively. D and E, β-galactosidase assay of yeast two-hybrid interactions of S. cerevisiae Fob1 and S. cerevisiae Fob1 mutant I407T with S. cerevisiae Net1 and S. cerevisiae Sir2, respectively. F, two-dimensional agarose gel analysis of replication intermediates from fob1Δ of YSB348 containing vector (pGAD424), pGAD424 S. cerevisiae FOB1, or pGAD424-FOB1I407T.

FIGURE 5.

Fob1-dependent silencing at the end of rDNA array does not require replication fork protection proteins. A, schematic representation of the end of the rDNA array showing the mURA3 cassette. B, silencing of the mURA3 cassette in wild type, sir2Δ, fob1Δ, tof1Δ, and fob1Δcsm3Δ strains. C, silencing of the mURA3 cassette in wild type, fob1Δ, and csm3Δ strains. The csm3Δ strain was derived by complementing a fob1Δcsm3Δ doubly deleted strain with a CSM3-expressing plasmid.

FIGURE 6.

Fob1-dependent silencing at the Ter sites in the middle of the rDNA array does not require replication fork protection proteins. A, a schematic view of the mURA3 silencing cassette cloned downstream of Ter sites in the middle of rDNA array. B, silencing of the mURA3 cassette occurs in both the WT and the tof1Δ strains. C, two-dimensional agarose gel analysis of replication intermediates showing abolition of fork arrest in the tof1Δ strain. D and E, ChiP assay showing binding of Fob1 to Ter sites in a tof1Δ strain. Ab, antibody; ARS, origin of replication.