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Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 9178-9188, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9178-9188.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
National Institute for Basic Biology and The Graduate University for Advanced Studies, School of Life Science, Okazaki 444-8585, Japan
Received 23 July 2003/ Returned for modification 8 September 2003/ Accepted 17 September 2003
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ABSTRACT |
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100-bp DNA sequence located near the 3' end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats. |
INTRODUCTION |
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In eukaryotic cells, replication fork-blocking sites have been identified in the rRNA gene repeats (rDNA) from yeast to human cells (for a review, see reference 41). They are called replication fork barriers (RFB) and inhibit replication forks in the direction opposite to rDNA transcription. The RFB has been studied most intensively in the yeast Saccharomyces cerevisiae. S. cerevisiae carries about 150 copies of the rDNA repeats. The RFB is located near the 3' end of the 35S rDNA in one of the nontranscribed spacer (NTS) regions (NTS1) (Fig. 1A). In the other NTS region (NTS2), there is an autonomously replicating sequence (ARS) which functions as a replication origin. In the S phase of the cell cycle, replication starts at the ARS bidirectionally and the rightward-moving replication forks are arrested at the RFB (Fig. 1). However, the other forks can go through the RFB site because of RFB polarity, like for Ter in E. coli (4, 29). Therefore, one of the biological roles of the RFB is thought to be as a barrier to prevent collision between replication and transcription machineries. Recently, we observed such a collision in an RFB-deficient mutant (44) (see Discussion). Ward et al. identified two closely spaced sequences, the RFB1 and RFB2 sites, in the RFB (47). These sites are responsible for the major and minor replication fork-blocking activities, respectively, that are seen in two-dimensional (2D) gel electrophoresis. We also identified a third site (here it is named RFB3) between the HindIII and EcoRI sites by using a strain whose RFB1 and RFB2 sites (HpaI-HindIII) are deleted (31) (for details, see Fig. 3 and 5). In this paper, I call these sites collectively the RFB site.
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In terms of rDNA recombination, the RFB site is thought to be a recombination hot spot that induces DNA double-strand breaks (31). FOB1 was shown to be essential for recombination in the rDNA (22, 23, 27, 36). FOB1-dependent recombination in the rDNA is known to be required for regulation of copy number, probably through unequal sister-chromatid recombination after a double-strand break at the RFB (27). Double-strand breaks also produce extrachromosomal rDNA circles, whose accumulation seems to be a cause of aging (42, 40). In fact, in a fob1 mutant, the life span is extended more than 50% compared to that of wild-type cells (6). As another aging gene, SIR2, whose gene product is a NAD-dependent histone deacetylase (20), is genetically downstream of FOB1 in the aging process, instability of the rDNA is probably an important component of this process (23).
We are interested in the molecular mechanisms of the RFB- and FOB1-dependent recombination and replication fork-blocking activity, and therefore in this study I aimed to resolve the enzymatic function of Fob1p. Perhaps the most likely function is RFB binding activity analogous to the action of Tus on the Ter sequence in E. coli. However, there are currently no reports showing that Fob1p binds to the RFB sequence. In this study, I demonstrate that Fob1p binds to the RFB and inhibits the replication fork in vivo and that this binding is characterized by an unusual wrapping activity.
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MATERIALS AND METHODS |
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The yeast strains and plasmids used are listed in Table 1. Disruption of FOB1 was described previously (30). Plasmid pTAK901 was constructed by inserting the PCR-amplified open reading frame (ORF) of FOB1 into the multicloning site of pESC-LEU (Stratagene). Point mutations were added to pTAK901 by site-directed mutagenesis (performed by Bio Dynamics Laboratory Inc., Tokyo, Japan) to construct pTAK991 to -995. pTAK900 was constructed by inserting the ORF of FOB1 into the BamHI-SalI site in pEG(KT), and glutathione S-transferase (GST) was added to the N terminus of Fob1p. Plasmids pTAK902.1 to -9 were constructed by inserting PCR products amplified from several regions around the RFB into the BamHI-SphI site of the YEp24 shuttle vector (New England Biolabs). pTAKc1 to -f4 were constructed by inserting annealed oligonucleotides into the BamHI-SphI site of YEp24. TAK899 was constructed by inserting a URA3 gene into the rDNA repeats.
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105 cells) with various FOB1 plasmids (pTAK991 to -995) was grown to stationary phase in SGal lacking uracil at 30°C. The frequencies of Ura- recombinants were then determined by spotting aliquots of 10-fold serial dilutions of the cultures onto SD with and without 5-fluoro-orotic acid. Purification of GST-Fob1p. Purification of GST-Fob1p was performed as described previously (33). In short, a 2-liter YK9 culture with pTAK900 was grown to mid-logarithmic phase at 30°C in SD lacking uracil. The cells were transferred to YPGal and incubated for 4 h at 30°C. The cells were then harvested, destroyed with glass beads, and applied to a GST affinity column (Amersham Pharmacia Biotech.). The eluted fraction was further purified by gel filtration, and the GST-Fob1p fraction was collected.
DNA binding assay. Gel shift assays were performed as follows. The RFB fragment (fragment 7 in Fig. 3B) and subfragments (70-bp DNA fragments, made by PCR with pTAKc1 to -f4 as the templates) which contain several parts (each 24 bp in length) of the RFB (see Fig. 5C) were used as the DNA target. These DNA fragments were end labeled with 32P and mixed with GST-Fob1p in 20 µl of phosphate buffer (20 mM sodium phosphate [pH 7.4], 1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 100 µg of bovine serum albumin per ml, 0.01% Triton X-100). The mixture was incubated at 30°C for 30 min. The DNA-Fob1p complex was visualized on 5% polyacrylamide gels at 4°C. Footprinting assays were performed with the SureTrack footprinting kit (Pharmacia) according to the manufacturer's instructions.
AFM imaging and analysis. Atomic force microscopy (AFM) imaging was performed by the Research Institute of Biomolecule Metrology Co., Ltd., Tsukuba, Japan. Three micrograms of GST-Fob1p and 500 ng of the RFB fragment were mixed in 0.5 ml of phosphate buffer (20 mM sodium phosphate [pH 7.4], 1 mM EDTA, 2 M NaCl). The mixture was incubated at 25°C for 60 min and dialyzed in 20 ml of phosphate buffer (20 mM sodium phosphate [pH 7.4], 1 mM EDTA, 100 mM NaCl, 5 mM MgCl2) for 12 h. The mixture was then applied to a freshly cleaved mica surface, followed by successive washings with water and subsequent drying under nitrogen gas. Imaging was carried out with a Digital Instrument Nanoscope III with a type E scanner in the tapping mode.
Other methods. The chromatin immunoprecipitation (ChIP) assay was based on methods described previously (45), except that Dynabead protein A was used (25). The RFB activity was analyzed by 2D gel electrophoresis as previously described (2). Determination of rDNA copy number was done as previously described (27).
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RESULTS |
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A zinc finger motif is required for Fob1p association with the RFB, inhibition of the replication fork, and recombination in the rDNA. Computer analysis (PHI-BLAST; National Center for Biotechnology Information) (7) predicted that Fob1p contains a zinc finger motif, which is known to bind to DNA. To examine whether this motif is essential for Fob1p association with the RFB, point mutations were made in the FOB1 ORF to disrupt the motif (C2H2) (Fig. 2A) and were expressed in a fob1 mutant. Expression of the mutated Fob1p was confirmed by Western blotting (data not shown). A ChIP assay similar to that described in the previous section was performed, and the results are shown in Fig. 2B. Fob1p association with the RFB was completely lost in the strains whose zinc finger motif is disrupted (Fig. 2B, lanes 3 to 6). In contrast, a control mutation in the integrase catalytic core-like structure (Fig. 2A), which was also predicted by computer analysis (PHI-BLAST; National Center for Biotechnology Information) (7), did not affect the association (Fig. 2B, lane 7).
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I also tested the effects of the mutations on rDNA recombination. A URA3 marker was integrated into the rDNA repeats, and the rate of loss was detected on a fluoro-orotic acid plate. The results are shown in Fig. 2D. Mutations in the zinc finger motif reduced the recombination rate to the level found in fob1 mutants. In contrast, the control mutation did not affect the rate (D291A). Taken together, these results suggest that the zinc finger motif of Fob1p is required for the association with the RFB and that this association is essential for both the replication fork-blocking and recombination activities at the RFB.
Fob1p associates with a broad region in the RFB in vivo to inhibit DNA replication. To study in detail the Fob1p-associated region in the RFB, nine fragments of the RFB and the surrounding region were cloned into the SphI-BamHI site of the YEp24 shuttle vector (Fig. 3A and B). As a plasmid-cloned RFB was shown to inhibit the replication fork (4, 29), association of Fob1p should be observed on active RFB fragments in the plasmid. The plasmids were transformed into the yeast strain NOY408-1bf, which has Fob1-FLAGp (pTAK901), and a ChIP assay was performed as outlined above. Two PCR primer sets for the detection of the precipitated fragments were designed (Fig. 3A). One amplifies a vector region as an internal control for the ChIP assay, and the other amplifies the subcloned RFB fragments. The results of the ChIP assay are shown in Fig. 3C (upper panel). There are two bands in the ethidium bromide-stained gel in each lane. The lower band is the vector control fragment, and the upper bands correspond to the subcloned RFB fragments. The intensities of the bands were measured, ratios of the upper to the lower bands were calculated, and the values were plotted on a graph (Fig. 3C, lower panel). The intensities of fragments 1, 3, 7, 8, and 9 were higher than those of other fragments. In contrast, when total DNA (whole-cell extract) was used as a template for the PCR, the ratios were similar (Fig. 3C, middle and lower panels). The results of the ChIP assay are summarized in Fig. 3B, where fragments which were associated with Fob1p are indicated. As shown in Fig. 3B, Fob1p associates with a broad region of DNA corresponding to the RFB site.
Next, I investigated the DNA replication fork-blocking activity of the subcloned RFB fragments by 2D analysis. The results are shown in Fig. 3D. Fragments 1, 3, 7, 8, and 9 showed replication fork-blocking activity, and these fragments correspond precisely to the Fob1p-associated fragments identified in Fig. 3C. Interestingly, two spots are observed within fragment 9, although only one spot was observed for fragment 7. This could be because part of RFB1 was deleted in fragment 9 and thus RFB1 activity was reduced, allowing the RFB2 and RFB3 activities to be detected. This suggests that RFB1 is the strongest inhibitor of the replication fork and that most replication forks arrest at RFB1. RFB2 and RFB3 are adjacent, and therefore their spots are expected to be fused. These results strengthen the conclusion that Fob1p association with the RFB site is required for the replication-blocking activity.
Fob1p specifically binds to the RFB in vitro. To examine whether Fob1p associates with the RFB itself or whether it requires other factors, I purified Fob1p for in vitro analysis. Fob1p with a GST tag was overexpressed in yeast, and GST-Fob1p was isolated with GST affinity column. A few contaminating bands were visible with Coomassie blue staining, and therefore the solution was treated by gel filtration to remove these bands. Figure 4C shows the purified GST-Fob1p separated by acrylamide gel electrophoresis and stained with Coomassie blue. As the fusion gene, GST-FOB1, could complement RFB and rDNA copy number expansion deficiencies in a fob1 mutant in vivo (Fig. 4A and B), I used the fusion protein for further in vitro analysis.
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Fob1p binds to two separate sequences in the RFB and interacts with several other sites. To identify precisely where in the RFB Fob1p binds, a DNase I footprinting assay was performed. GST-Fob1p was mixed with the RFB fragment (fragment 7 in Fig. 3B), and this complex was partially digested by DNase I, followed by denaturing gel electrophoresis. In the left panel of Fig. 5A, the 5' end of the RFB fragment close to the HindIII site was labeled with 32P, and in the right panel, the other end was labeled. Therefore, protection of both strands by Fob1p against DNase I was determined. As shown in the left panel of Fig. 5A, apparent protection was detected in the F1 region, which corresponds to RFB1 (Fig. 5C). Beside F1 is a DNase I-hypersensitive site, which are often observed near protein binding sites. In regions F2 to F4, the protection is not so clear, but some degree of protection is observed. F3 and F4 are located in RFB2 and RFB3, respectively (Fig. 5C).
To investigate the binding activity of each region more precisely, I performed gel shift assays with small fragments (24 bp) from the RFB site, surrounded by nonyeast DNA. Fragments f1 to f4 and the controls (c1 and c2, which are regions flanking the RFB [Fig. 5C]) were cloned into the SphI-BamHI site of YEp24 (Fig. 3A), and PCR amplicons including the 24-bp sequences were amplified. The fragments were end labeled with 32P and used as substrates for the gel shift assay. The results are shown in Fig. 5B. GST-Fob1p binds to f1 and f4. However, binding affinities of the f2 and f3 fragments are at the same level as the control (c1 and c2). Fragment f1 showed about three times stronger binding affinity to Fob1p than f4 did. I conclude that GST-Fob1p independently binds to these two regions, which correspond to RFB1 and RFB3.
The small 24-bp fragments still have replication fork-blocking activity. 2D analysis was used to examine the replication fork-blocking activity of the small, 24-bp fragments as in Fig. 3D. The results are shown in Fig. 6. Although the intensities are not as strong as those in Fig. 3D, spots corresponding to the accumulation of Y-shaped replication intermediates arrested at the inserted fragments were detected in f1 and f4. Fragment f1 showed about three times stronger blocking activity than f4, corresponding to the results of the shift assay. These results further demonstrate that replication fork-blocking activity is associated with Fob1p binding activity and that binding to one region alone is sufficient for replication fork-blocking activity.
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90 bp) shorter than that of the original (Fig. 7C). This length corresponds to the distance between RFB1 and RFB3. This further suggests that in these complexes, the DNA wraps around Fob1p (Fig. 7D, model b). Therefore, the RFB sequence may wrap around Fob1p in vivo, and this structure might be important for function (see Discussion).
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DISCUSSION |
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100 bp). I speculate that this wrapping structure is important for stable binding to a silenced locus by behaving like a nucleosome. Defossez et al., using immunofluorescence microscopy, first reported the nucleolar localization of Fob1p (6). More recently, Gadal et al., using microscopy, also observed that Fob1p was located at the nucleolar-nucleoplasmic interface with the rDNA (11). DNA binding activity of Fob1p was also suggested by the discovery of a zinc finger motif, which often contributes to DNA binding activity (7). Here I show that this motif is responsible for the association (Fig. 2B). Dlakic predicted the 3D structure of Fob1p and pointed out the similarity of a domain to the integrase catalytic core-like structure of retrotransposons (Fig. 2A) (7). I mutated the conserved asparatic acid of this core structure to alanine (D291A) (Fig. 2A) to see the effect on the function of Fob1p. As shown in Fig. 2D, the mutation was not important, at least for DNA binding, replication fork-blocking, and recombination activities in the rDNA. Therefore, the predicted integrase catalytic core-like structure may have another, unknown function.
We previously reported that there is a 69-bp minimum essential sequence required for inhibition of the replication fork with the plasmid-cloned RFB (29). However, here I demonstrate that a 24-bp sequence (f1 in Fig. 6, corresponding to RFB1) from the original 69-bp sequence is enough to arrest the fork. In addition, I found another replication fork-blocking activity outside the 69-bp sequence (f4 in Fig. 6, corresponding to RFB3). One reason for the increased sensitivity in detecting blocking activity in this system is that Fob1p is overexpressed by the galactose-inducible promoter. As there are 150 copies of the rDNA, each with an RFB site, with wild-type levels of Fob1p there might be not enough Fob1p to inhibit the replication forks on all of the RFB plasmids. Ward et al. identified two regions (RFB1 and RFB2) responsible for replication fork-blocking activity as two separated spots in a 2D analysis (47). RFB3 was identified by deletion analysis in a strain with only two copies of the rDNA (31). After deletion of HpaI-HindIII fragment which contains RFB1 and RFB2, weak replication fork-blocking activity (RFB3) still remains near the HindIII site. Ward et al. pointed out that in addition to the RFB2 sequence, an inverted repeat region (IR1 and IR2 in Fig. 5C) overlapping RFB3 is also required for the second spot in 2D analysis (47). Brewer et al. also noted the importance of the HindIII-EcoRI fragment (Fig. 1A), which includes RFB3, for the RFB activity (4). Here I show that a 24-bp sequence, corresponding to RFB3, is capable of binding to Fob1p and inhibiting the replication fork. However, I detected neither RFB activity nor Fob1p association of the RFB2 fragment, although RFB2 did interact with Fob1p in the footprinting assay (Fig. 5A). From these data, I speculate that the second spot observed in the 2D analysis (4, 47) (Fig. 3D) is the sum of RFB2 and RFB3 activities and that for the activity of RFB2, Fob1p association with RFB3 is necessary. This association makes it possible that RFB2 interacts with Fob1p to inhibit replication. The reason that the second spot disappeared with modification of the RFB2 sequence (47) may be that RFB3 by itself is not strong enough to be detected in their system.
We hypothesize that Fob1p acts as a barrier to avoid collision between the replication and transcription machineries. Recently, we detected such a collision in a fob1 derivative strain whose rDNA copy number is reduced to 20 (44). In this strain, most of the rDNA copies are expected to be transcribed (9), and the collision occurs frequently enough to be detected as a region of slow replication fork progression by 2D analysis. Such collision may impose a burden by inducing damage in the rDNA. Actually, the stability of the rDNA was reduced, and recombination frequently took place despite FOB1 deficiency in this strain. Recombination repair, which includes new DNA synthesis, may result in errors more frequently than normal DNA replication (43). Therefore, the RFB function may have evolved to prevent harmful mutations that result from collision (44).
In this study I demonstrated that Fob1p binds to the RFB. However, the molecular mechanism by which this complex inhibits the replication fork is still unknown. I speculate that one possible target of Fob1p may be DNA helicases, similar to the action of Tus in E. coli (1, 13, 32), although there is no homology between Fob1p and Tus. Ivessa et al. identified two DNA helicases (Rrm3p and Pif1p) which appear to function in rDNA replication (21). In an RRM3 mutant, replication stops at several sites in the rDNA. Moreover, the RFB activity doubles in the mutant. However, in a PIF1 mutant, the RFB activity decreases about threefold. Although the mechanism by which the helicases influence RFB activity is unknown, it is possible that their action is a target for Fob1p.
The binding action of Fob1p is unusual. AFM images appear to show the RFB sequence wrapped around the protein (Fig. 7D, model b). The distance between RFB1 and RFB3 (90 bp) suggests that Fob1p may act as a dimer in this wrapping structure, although it is difficult to know the circumference of Fob1p from its molecular weight. The binding activity of Fob1p was eliminated by disruption of the zinc finger motif, and therefore the motif seems to bind both RFB1 and RFB3 independently. This is consistent with Fob1p activity as a dimmer. The results in Fig. 3D and 6 suggest that RFB1 provides the strongest inhibition of the replication fork (Fig. 7D, model c). This is supported by its stronger binding activity to Fob1p in vitro (Fig. 5B). Therefore, the DNA binding site of Fob1p has different affinities for RFB1 and RFB2. Determination of the crystal structure of the protein will help reveal the precise structure of the Fob1p-RFB complex. It is noted that the GST tag fused to Fob1p is known to dimerize proteins. Therefore, there is a possibility that the tag induces dimerization, thus causing the RFB sequence to wrap around the GST-Fob1p dimer. However, the existence of adjacent RFB1 and RFB3 sites suggests that dimerization of Fob1p occurs in nature.
This wrapping structure itself is not essential for the RFB activity in the plasmid system, because small RFB fragments (24 bp of RFB1 or RFB3) inhibited the replication fork (Fig. 6). However, in the rDNA, the wrapping structure may be important for function. The RFB region is known to be silenced by Sir2p, where the chromatin structure becomes condensed (10). In this environment, the wrapping structure may be helpful for Fob1p to access the silenced chromatin structure and stay there. As shown in Fig. 1B, Fob1p association in the rDNA is detectable by ChIP assay without cross-linking. This indicates that the association is stably maintained during the high-salt washing process. Therefore, I speculate that by increasing the length of associating region, the wrapping nucleosome-like structure may allow Fob1p to bind the DNA and be stably maintained in the rDNA chromatin. Nucleosome mapping (48) around the RFB by using micrococcal nuclease indicated that nucleosomes are located around the RFB region, and, interestingly, the RFB site was also protected from the nuclease (unpublished data). This pattern of protection is consistent with a nucleosome-like wrapping structure of the Fob1p-RFB complex.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants-in-aid for scientific research 13141205, 13480234, and 14380332 from the Ministry of Education, Science and Culture, Japan; by a grant from the Ministry of Health and Welfare, Japan; and by a grant from the Human Frontier Science Organization, France.
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FOOTNOTES |
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