The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication
2003; Springer Nature; Volume: 22; Issue: 8 Linguagem: Inglês
10.1093/emboj/cdg180
ISSN1460-2075
AutoresGwennaëlle Versini, Itys Comet, Michelle Wu, Laura L. Mays Hoopes, Étienne Schwob, Philippe Pasero,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoArticle15 April 2003free access The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication Gwennaelle Versini Gwennaelle Versini Institute of Molecular Genetics, CNRS and Université Montpellier II, 1919 route de Mende, 34293 Montpellier, France Search for more papers by this author Itys Comet Itys Comet Present address: Institute of Human Genetics, CNRS, 141 Rue de la Cardonille, 34396 Montpellier, France Search for more papers by this author Michelle Wu Michelle Wu Biology Department and Molecular Biology Program, Pomona College, Claremont, CA, 91711 USA Search for more papers by this author Laura Hoopes Laura Hoopes Biology Department and Molecular Biology Program, Pomona College, Claremont, CA, 91711 USA Search for more papers by this author Etienne Schwob Etienne Schwob Institute of Molecular Genetics, CNRS and Université Montpellier II, 1919 route de Mende, 34293 Montpellier, France Search for more papers by this author Philippe Pasero Corresponding Author Philippe Pasero Institute of Molecular Genetics, CNRS and Université Montpellier II, 1919 route de Mende, 34293 Montpellier, France Search for more papers by this author Gwennaelle Versini Gwennaelle Versini Institute of Molecular Genetics, CNRS and Université Montpellier II, 1919 route de Mende, 34293 Montpellier, France Search for more papers by this author Itys Comet Itys Comet Present address: Institute of Human Genetics, CNRS, 141 Rue de la Cardonille, 34396 Montpellier, France Search for more papers by this author Michelle Wu Michelle Wu Biology Department and Molecular Biology Program, Pomona College, Claremont, CA, 91711 USA Search for more papers by this author Laura Hoopes Laura Hoopes Biology Department and Molecular Biology Program, Pomona College, Claremont, CA, 91711 USA Search for more papers by this author Etienne Schwob Etienne Schwob Institute of Molecular Genetics, CNRS and Université Montpellier II, 1919 route de Mende, 34293 Montpellier, France Search for more papers by this author Philippe Pasero Corresponding Author Philippe Pasero Institute of Molecular Genetics, CNRS and Université Montpellier II, 1919 route de Mende, 34293 Montpellier, France Search for more papers by this author Author Information Gwennaelle Versini1, Itys Comet2, Michelle Wu3, Laura Hoopes3, Etienne Schwob1 and Philippe Pasero 1 1Institute of Molecular Genetics, CNRS and Université Montpellier II, 1919 route de Mende, 34293 Montpellier, France 2Present address: Institute of Human Genetics, CNRS, 141 Rue de la Cardonille, 34396 Montpellier, France 3Biology Department and Molecular Biology Program, Pomona College, Claremont, CA, 91711 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1939-1949https://doi.org/10.1093/emboj/cdg180 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity. RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers. The human BLM and WRN helicases are required for normal S phase progression. In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild-type kinetics. To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1Δ cells. Unexpectedly, we find that these cells progress faster through S phase than their wild-type counterparts. Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase. However, completion of DNA replication is strongly retarded at the rDNA array of sgs1Δ cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus. These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase. Introduction The SGS1 gene of Saccharomyces cerevisiae encodes a DNA helicase of the RecQ family, which is involved in the maintenance of genome integrity in all organisms analyzed to date, from bacteria to human (Karow et al., 2000). Sgs1p is closely related to the human helicases BLM, WRN and RECQL4, defective in Bloom syndrome (BS), Werner syndrome (WS) and Rothmund–Thomson syndrome (RTS), respectively. These diseases are characterized by chromosome instability, hyper-recombination and an increased incidence of cancers (Mohaghegh and Hickson, 2001). However, the molecular events underlying these phenotypes have remained obscure. New insights into the molecular basis of these disorders came with the finding of functional interactions between RecQ helicases and multiple components of the replication machinery, including proliferating cell nuclear antigen (PCNA), RP-A, topoisomerases and DNA polymerase δ (Gangloff et al., 1994; Watt et al., 1995; Lebel et al., 1999; Brosh et al., 2000; Wu et al., 2000). In yeast and human cells, levels of RecQ helicases peak in S phase (Dutertre et al., 2000; Frei and Gasser, 2000), and these enzymes were shown to co-localize with sites of ongoing DNA synthesis in yeast and in Xenopus (Frei and Gasser, 2000; Chen et al., 2001). Moreover, cells derived from BS and WS patients accumulate abnormal replication intermediates (Lonn et al., 1990; Poot et al., 1992). Taken together, these data argue for a role for RecQ helicases at the replication fork. Unlike components of the MCM complex—the presumed helicase of the growing fork (Labib and Diffley, 2001)—RecQ proteins are not essential for cell growth in the budding and fission yeasts and are therefore not likely to be involved in processive DNA synthesis. The simultaneous inactivation of Sgs1p and Srs2p, another DNA helicase, induces major growth defects in S.cerevisiae (Gangloff et al., 2000; McVey et al., 2001), and it had been proposed initially that Sgs1p and Srs2p are required for replication fork progression (Lee et al., 1999). However, this low viability of sgs1 srs2 cells is suppressed by the inactivation of the homologous recombination pathway, demonstrating that Sgs1p is not required for processive DNA synthesis, but rather is involved in the suppression of inappropriate recombination in S phase (Gangloff et al., 2000). Interactions between RecQ helicases and components of the DNA recombinational repair machinery further support a role for these enzymes in the coordination of replication and recombination. In mammals, BLM is a component of BASC, a genome surveillance complex coordinating multiple recombination and repair activities (Wang et al., 2000). Like BLM, the yeast Sgs1 helicase interacts with Rad51p and Top3p (Bennett et al., 2000; Wu et al., 2000, 2001). Moreover, Sgs1p functions as a sensor in the intra-S phase checkpoint, potentially linking DNA recombination to cellular surveillance mechanisms (Cobb et al., 2002; Kolodner et al., 2002). The partial complementation of the hyper-recombination phenotype of the yeast sgs1Δ mutant by the expression of either BLM or WRN suggests that their function has been conserved from yeast to human (Yamagata et al., 1998). However, RecQ activity seems to be dispensable for S phase progression in yeast, and sgs1Δ cells grow with wild-type kinetics. In contrast, cells derived from BS and WS patients display a prolonged S phase, with slow fork progression and accumulation of abnormal replication intermediates (Hand and German, 1975; Lonn et al., 1990; Poot et al., 1992). Whether this difference reflects a specialization of RecQ function in higher eukaryotes or whether it indicates that the same function is differentially required for S phase progression in yeast and mammals remains to be addressed. Here, we have monitored S phase progression in wild-type and sgs1Δ yeast cells using flow cytometry, pulsed-field gel electrophoresis (PFGE), two-dimensional gel analysis and a new technique called DNA combing, which allows the analysis of replication on single DNA molecules. We show that sgs1Δ cells traverse S phase significantly faster than wild-type cells because forks are moving more rapidly. In contrast, we find that completion of DNA replication is impaired at ribosomal DNA (rDNA), which contains a high density of replication fork barriers. These data suggest that Sgs1p is required neither for elongation nor for the decatenation of newly replicated chromosomes, but plays an important role in the suppression of illegitimate recombination occurring at stalled replication forks. Results The Sgs1 helicase slows down progression through S phase To examine the effect of SGS1 inactivation on DNA replication, congenic wild-type (E1000) and sgs1Δ (E1245) cells were arrested in G1 with α-factor and released synchronously into the cell cycle. Cells were collected every 15 min from release, and DNA content was monitored by flow cytometry (Figure 1A). The appearance of buds, indicative of entry into the cell cycle, was scored (Figure 1B). Surprisingly, the DNA content appeared to increase faster in the sgs1Δ mutant than in wild-type cells (Figure 1A; compare time points 30 and 45 min), although both strains passed START synchronously (Figure 1B). In order to quantify this difference, the area of S phase peaks at t = 30 min was integrated in four independent experiments (example in Figure 1C). The DNA content per haploid cell was 36% (± 8%) greater at this time in the absence of Sgs1 helicase. Interestingly, flow cytometry analysis of log phase cultures grown on synthetic complete medium at 25°C revealed that the proportion of cells with a 2C DNA content is ∼40% higher in the sgs1Δ mutant (Figure 1D), indicating that although sgs1Δ cells progress faster through S phase, they accumulate transiently in G2 or M phase. Figure 1.S phase progression is accelerated in sgs1Δ cells. (A–C) Wild-type (E1000) and sgs1Δ (E1245) cells were arrested in G1 with α-mating factor and released synchronously into S phase. (A) Samples were collected at the indicated times after release and DNA content was analyzed by flow cytometry. (B) Budding index. (C) Flow cytometry profiles of wild-type and sgs1Δ cells collected at t0 (G1) and t30 min (early S). The proportion of the area of the t30 min profile extending beyond the G1 peak is 42% larger in sgs1Δ mutants than in wild-type cells. (D) Flow cytometry analysis of exponentially growing wild-type and sgs1Δ cells. The proportion of G2/M cells is 40% (± 6%) higher in the sgs1Δ mutant. Download figure Download PowerPoint Sgs1 is not required for the termination of DNA replication Sgs1p binds topoisomerases II and III, which are required for the decatenation of late replication intermediates (Gangloff et al., 1994; Watt et al., 1995). The elevated rate of missegregation in sgs1Δ cells could therefore reflect their inability to resolve newly replicated chromosomes. To test this hypothesis, we have monitored the completion of DNA replication in wild-type and sgs1Δ cells by PFGE. This approach is based on the fact that incompletely replicated chromosomes cannot be separated by PFGE (Hennessy et al., 1991). Thymidine kinase-expressing (TK+) cells, which are capable of incorporating bromodeoxyuridine (BrdU) (Lengronne et al., 2001), were released synchronously from a G1 arrest in complete medium supplemented with BrdU and were collected at regular intervals throughout the cell cycle (Figure 2A and B). Chromosomal DNA from wild-type and sgs1Δ cells was subsequently purified in agarose plugs and was separated by PFGE. As expected, BrdU-labeled DNA did not enter the gel until the end of S phase (Supplementary figure 1 available at The EMBO Journal Online). The re-emergence of fully replicated chromosomes was quantitated after transfer to a nitrocellulose membrane and detection of BrdU (Figure 2C and D). Interestingly, the bulk of chromosomal DNA re-entered the gel 5–10 min earlier in sgs1Δ cells, which represents approximately a quarter of the length of S phase in wild-type cells. This faster completion of DNA replication is reminiscent of the accelerated S phase progression observed by flow cytometry. Moreover, it shows that Sgs1p is not required for the timely resolution of late replication intermediates. However, the transient accumulation of cells with a 2C DNA content (Figure 1D) suggests that, although the bulk of genomic DNA is rapidly replicated, a significant proportion of the cells may experience problems in separating their chromosomes. Figure 2.The Sgs1p helicase is not required for the completion of DNA replication. Wild-type (E1000) and sgs1Δ (E1245) cells were released synchronously from an α-factor block in medium supplemented with 400 μg/ml BrdU and harvested at the indicated times. (A and B) Analysis of DNA content by flow cytometry. (C) Genomic DNA prepared from cells embedded in low-melting agarose plugs was separated by PFGE. The amount of BrdU incorporated in fully replicated chromosomes was quantitated as described in Materials and methods. (D) Kinetics of the re-emergence of fully replicated chromosomes in wild-type and sgs1Δ cells. Relative BrdU incorporation was quantitated for six representative chromosomes. Error bars indicate standard deviation. Download figure Download PowerPoint Faster S phase progression in sgs1Δ cells is not due to a premature G1/S transition To check whether the faster completion of DNA replication in sgs1Δ cells is due to a premature entry into S phase, we have analyzed the kinetics of origin activation on a specific chromosome (chromosome III) in wild-type and sgs1Δ cells by PFGE (Figure 3A and B). In both strains, chromosome III is excluded progressively from the gel 10 min after release, indicating that cells enter synchronously into S phase. In contrast, chromosome III mobility decreased only after 40 min in clb5Δ clb6Δ cells (Figure 3B), which is consistent with the fact that S phase is delayed by 30 min in this mutant (Schwob and Nasmyth, 1993). Analysis of other yeast chromosomes gave the same result (data not shown). To confirm this observation using an independent approach, we have examined the time of ARS306 activation in wild-type and sgs1Δ cells by two-dimensional gel electrophoresis (Brewer and Fangman, 1987). ARS306 is the first origin to fire on chromosome III. As shown in Figure 3C, it is activated ∼20 min after release from the α-factor arrest in both strains. However, we noticed that replication intermediates disappeared more rapidly in the sgs1Δ mutant, suggesting either that initiation is more synchronous in this population of cells or that forks progress faster in the absence of Sgs1p. Interestingly, the time of ARS306 activation corresponds exactly to the time of the exclusion of chromosome III from the gel. However, this chromosome completed replication faster in sgs1Δ cells (Figure 3B), which is consistent with the data shown in Figure 2D. Taken together, these results indicate that the faster replication in sgs1Δ cells is not due to premature entry into S phase. Figure 3.Time of origin firing in wild-type and sgs1Δ cells. (A) Wild-type (E1000), sgs1Δ (E1245) and clb5Δ clb6Δ (E742) cells were arrested in G1 with α-factor. Cells were collected every 10 min after degradation of α-factor, and DNA content was analyzed by flow cytometry. (B) The electrophoretic mobility of chromosome III was analyzed by PFGE, and DNA content was quantitated with a phosphoimager. (C) Analysis of the timing of origin activation in wild-type (E1000) and sgs1Δ (E1245) cells. Cells were released synchronously into S phase from an α-factor block, and samples were taken at the indicated times after release. Neutral/neutral two-dimensional gel analysis (Brewer and Fangman, 1987) was used to monitor initiation at the early replication origin ARS306 and at the late origin ARS501. (D) Flow cytometry profiles of the samples analyzed in (C). (E) Two- dimensional gel analysis of initiation at the early origin ARS306, the late origin ARS603 and the dormant origin ARS301 in wild-type (E1000), sgs1Δ (E1245) and rad53-11 (E1019) cells released into S phase for 90 min in the presence of 200 mM HU. Download figure Download PowerPoint The mechanisms delaying initiation at late origins are functional in sgs1Δ cells We next analyzed the time of initiation at the subtelomeric late origin ARS501 and the internal late origin ARS603 using two-dimensional gel electrophoresis. We found that these origins are activated ∼10 min after the early origin ARS306 in both strains (Figure 3C, and data not shown). Although late origins appeared to fire slightly earlier in a fraction of sgs1Δ cells, this suggests that the timing of origin activation is not significantly affected by the absence of Sgs1p. Late origins are normally repressed by the replication checkpoint in the presence of hydroxyurea (HU) (Santocanale and Diffley, 1998), but fire in rpd3Δ cells exposed to HU, presumably because late origin initiation is advanced in these cells (O.Aparicio, unpublished results). To check whether this is also the case for sgs1Δ cells, we analyzed the activity of the late origin ARS603 and of the dormant origin ARS301 by two-dimensional gel electrophoresis. As shown in Figure 3E, these origins are repressed in wild-type and in sgs1Δ cells, but fire in the rad53-11 mutant (Santocanale and Diffley, 1998; Santocanale et al., 1999). Similarly, ARS501 is inactive in sgs1Δ cells exposed to HU (J.Cobb and S.Gasser, submitted). Taken together, these results indicate that the mechanisms controlling the activation of late replication origins are active in sgs1Δ cells. The accelerated S phase in sgs1Δ cells is not due to a higher frequency of initiation Several lines of evidence indicate that most of the yeast replication origins are active in only a fraction of the cell cycles. In sgs1Δ cells, an overall increase of this initiation rate could explain their faster progression through S phase. To address this possibility, we have compared the density in active origins in wild-type and sgs1Δ cells using BrdU incorporation and dynamic molecular combing (Michalet et al., 1997). Cells were released synchronously into S phase in the presence of BrdU to label initiation sites, and elongation was blocked with the addition of HU (Figure 4A). Chromosomal DNA was prepared in agarose plugs to avoid shearing and was combed on silanized coverslips. The sites of BrdU incorporation were subsequently detected along individual molecules with fluorescent antibodies, and DNA fibers were counterstained with an anti-guanosine antibody (Figure 4C). Since combed DNA fibers are stretched uniformly (Michalet et al., 1997), the statistical analysis of center to center distances between adjacent BrdU tracks provides an accurate indication of origin density (Lengronne and Schwob, 2002; Pasero et al., 2002). Here, the analysis of ∼10 Mb of DNA per strain revealed that active origins are not more abundant in sgs1Δ cells than in wild-type cells (Figure 4B). It is worth noting that initiation at late and dormant origins is not detected in this assay. However, we confirmed by two-dimensional gel electrophoresis that there is no promiscuous activation of dormant origins such as ARS301 in untreated sgs1Δ cells (data not shown). We therefore believe that the faster S phase of sgs1Δ cells is not due to an overall increase of origin activity. Figure 4.Single molecule analysis of the initiation rate in wild-type and sgs1Δ cells. (A) Wild-type (E1000) and sgs1Δ (E1245) cells were synchronized in G1 with α-factor and were released for 90 min into S phase in the presence of BrdU and 200 mM HU, to block elongation. (B) DNA fibers were combed on silanized coverslips and the center to center distances between adjacent BrdU tracks were measured as described in Materials and methods. For each strain, a total of 9.4 Mb of DNA was analyzed. Means and standard deviations are indicated. (C) Examples of DNA fibers. The center to center distance between BrdU tracks is indicated in kbp. Green, BrdU; red, DNA. Download figure Download PowerPoint Progression of replication forks is accelerated in sgs1Δ cells An alternative explanation for the faster S phase in sgs1Δ cells could be that forks are moving more rapidly in the absence of the Sgs1p helicase. In the experiment described above, we found that BrdU tracks were on average 22% longer in HU-arrested sgs1Δ cells (wild type, 18.4 ± 9.2 kb; sgs1Δ, 22.5 ± 12.2 kb). To examine whether BrdU tracks are also longer in sgs1Δ cells in the absence of drug, wild-type and sgs1Δ cells were released synchronously from G1 and genomic DNA was isolated from cells harvested in mid-S phase (t = 30 min). Individual chromosomes were combed, and newly replicated regions were visualized using anti-BrdU antibodies (Figure 5A). The analysis of a large number of BrdU tracks (∼400 per strain) revealed that newly replicated stretches are 48% longer in sgs1Δ cells (Figure 5B). These data suggest that the rate of elongation is faster in sgs1Δ cells. Figure 5.Replication forks progress faster in the absence of Sgs1p. (A) Wild-type (E1000) and sgs1Δ (E1245) cells were released synchronously from an α-factor block in BrdU-supplemented medium as described in Figure 4, but this time HU was omitted. Samples were collected in mid-S phase (30 min after release) and BrdU tracks were detected after DNA combing. The length of BrdU tracks (kb) is indicated for two representative molecules. (B) Size distribution of BrdU tracks in wild-type and sgs1Δ cells. For each strain, ∼400 signals, encompassing >15 Mb in total, were measured. (C) Early log phase cultures of wild-type and sgs1Δ cells were labeled for 25 min with 400 μg/ml BrdU. The distribution of BrdU track length was determined as described above. For each strain, ∼300 BrdU signals were measured (>10 Mb in total). (D) Early log phase cultures of wild-type, sgs1Δ and rad53-11 (E1019) cells were labeled for 22 min and the distribution of BrdU tracks length was determined (400 BrdU signals for each strain). (E) The analysis of four independent sets of experiments indicates that BrdU tracks are reproducibly longer (43.8 ± 5.9%) in the absence of the Sgs1 helicase. Download figure Download PowerPoint To ensure that longer BrdU tracks in sgs1Δ cells are not due to an artifact of synchronization, exponentially growing cultures of wild-type and sgs1Δ cells were pulse labeled for 25 min with BrdU and the length of newly replicated segments was measured by DNA combing. Again, we found a 40–50% increase of the mean BrdU track length in sgs1Δ cells (Figure 5C and D). The combination of four independent experiments confirmed that BrdU tracks are 44% (± 6%) longer in sgs1Δ cells (Figure 5E). Taken together, these results indicate that the Sgs1p helicase is required neither for the progression of the replication fork nor for the timely resolution of replicated chromosomes. Instead, we find that Sgs1p slows down the global rate of elongation. The checkpoint kinase Rad53 does not slow down fork progression In response to fork arrest induced by genotoxic drugs, yeast cells activate the checkpoint kinases Mec1p and Rad53p, which in turn block late origin firing and stabilize stalled forks (Lopes et al., 2001; Tercero and Diffley, 2001; Sogo et al., 2002). Since Sgs1p is required for the activation of Rad53p in HU-arrested cells, at least in Rad24-deficient cells (Frei and Gasser, 2000), we asked whether Sgs1p acts through Rad53p to slow down fork progression in a normal S phase. If this were the case, a rad53 mutant should also display an accelerated fork progression in our assay. To test this possibility, we measured the length of BrdU tracks in congenic wild-type (E1000), sgs1Δ (E1245) and rad53-11 (E1019) cells pulse labeled in mid-log phase as described above. We found that BrdU tracks were roughly identical in wild-type and rad53 cells, while they were again 50% longer in sgs1Δ cells (Figure 5D). These data indicate therefore that Rad53p does not regulate the rate of elongation in unchallenged growth conditions. Completion of DNA replication is delayed at the rDNA locus of sgs1Δ cells Sgs1-deficient cells are sensitive to sublethal doses of HU, presumably because they fail to stabilize stalled forks (J.Cobb and S.Gasser, submitted). We therefore reasoned that a locus naturally containing a high density of replication fork barriers, namely the rDNA array (Brewer and Fangman, 1988; Linskens and Huberman, 1988), should be more prone to replication fork collapse than bulk genomic DNA. To test this possibility, the rDNA array was excised by restriction digestion (Supplementary figure 2A), and completion of rDNA replication was monitored by PFGE. In wild-type cells, we found that the rDNA array re-entered the gel slightly after genomic DNA, suggesting that branched structures persist for a longer period of time at this locus (Figure 6B). Interestingly, this delay was exacerbated in sgs1Δ cells, the rDNA array re-entering the gel 20–30 min after the other chromosomes (Figure 6B). The same delay was observed when the mobility of rDNA arrays from wild-type and sgs1Δ cells were compared with each other (Supplementary figure 2B and C), or when chromosome XII, which bears the rDNA array, was compared with other chromosomes (Supplementary figure 2F). Interestingly, a similar alteration of chromosome XII mobility, which bears the rDNA array, has been reported recently in sgs1-ts slx4Δ double mutants (Kaliraman and Brill, 2002). Figure 6.Altered electrophoretic mobility of the rDNA array in sgs1Δ cells. Wild-type (E1000), sgs1Δ (E1245), rad52Δ (E1384) and sgs1Δ rad52Δ (E1382) cells were released synchronously into S phase in the presence of 400 μg/ml BrdU, and samples were collected at the indicated times. (A) Analysis of DNA content by flow cytometry. (B) Comparative analysis of the electrophoretic mobility of genomic DNA (gDNA) and rDNA in the four different strains. The re-emergence of fully replicated DNA molecules was quantitated as described in Figure 2. Download figure Download PowerPoint Wild-type and sgs1Δ cells display similar RFB and ARS activities The rDNA of S.cerevisiae contains ∼150 ARS elements, but only 20% of these potential replication origins are used every cell cycle. Forks moving leftward from the rDNA origin are arrested by a structure called a replication fork barrier (RFB), which persists until it merges with rightward-moving forks (Brewer and Fangman, 1988; Linskens and Huberman, 1988). To check if the altered mobility of the rDNA in sgs1Δ cells was due to a delayed initiation at this locus or to a persistence of stalled forks in late S phase, we have monitored the activity of the rDNA ARS and of the RFB by two-dimensional gel electrophoresis. The quantitation of replication intermediates in three independent experiments showed that origin activity and RFB signals were virtually identical in exponentially growing wild-type and sgs1Δ cells (Figure 7A). To confirm this result, we have analyzed rDNA replication in cells released synchronously in S phase. Bubble arcs were detected throughout the S phase at the rDNA (Figure 7B). Quantitation of RFB signals with a phosphoimager revealed a similar pattern in both strains, although the rDNA replicated slightly earlier in the sgs1Δ mutant (Figure 7C; Supplementary figure 3A). Moreover, the amount of stalled forks decreased after 50 min in both strains, and RFB signals were almost undetectable in sgs1Δ cells 20 min later (Supplementary figure 3B). We therefore assume that the altered electrophoretic mobility of the rDNA in sgs1Δ cells is not due to a replication initiation defect nor to the persistence of stalled replication forks. Figure 7.Analysis of rDNA replication and RFB activity by two-dimensional gel electrophoresis. (A) Genomic DNA was extracted from exponentially growing wild-type (E1000) and sgs1Δ (E1245) cells, and a StuI–XbaI rDNA fragment (3.5 kb) centered around the origin was analyzed by two- dimensional gel electrophoresis. Quantitation of bubble arc (open arrowhead) and RFB signals (replication fork barrier, filled arrowhead) with a phosphoimager did not reveal any significant difference between the two strains in three independent experiments. (B) Two-dimensional gel analysis of rDNA ARS and RFB activity in wild-type and sgs1Δ cells released synchronously into S phase (see Figure 3D for the corresponding flow cytometry profiles). (C) Quantitation of RFB signals in wild-type and sgs1Δ cells. Download figure Download PowerPoint The altered mobility of the rDNA array is due to homologous recombination Deletion of the SGS1 gene increases homologous recombination at rDNA (Gangloff et al., 1994). To check whether the altered electrophoretic mobility of this locus in sgs1Δ cells is due to the presence of unresolved recombination intermediates, we have inactivated the homologous recombination pathway in these cells. Interestingly, the migration of the rDNA array was restored in rad52Δ sgs1Δ cells (Figure 6B). In the absence of Sgs1p, the abnormal mobility of the rDNA array in late S phase is therefore likely to be attributable to the persistence of unresolved recombination intermediates. It is worth noting that we were unable to detect these intermediates on two-dimensional gels (Figure 7B). In contrast, a strong X-spike, corresponding to Holliday junctions, has been reported at the rDNA of polymerase α and δ mutants (Zou and Rothstein, 1997). We assume
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