A redundancy of processes that cause replication fork stalling enhances recombination at two
distinct sites in yeast rDNA
M. D. Mayán-Santos, M. L. Martínez-Robles, P. Hernández, J. B. Schvartzman*, D. B. Krimer
Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9,
28040 Madrid, Spain.
*E-mail schvartzman@cib.csic.es; Tel. (+34) 91 837 3112 ext. 4232; Fax (+34) 91 536 0432.
Introduction
The mechanisms involved in the maintenance of homogeneity within repeated families of DNA have attracted the
attention of scientists for many years (Smith, 1976; Coen et al., 1982; Nagylaki and Petes, 1982; Hillis et al., 1991;
Schlotterer and Tautz, 1994). It was early recognized that this phenomenon occurs via DNA recombination.
Homogenization of ribosomal DNA (rDNA) repeats is one of the best-characterized systems (Kobayashi et al., 1998 and
references therein; Kobayashi and Ganley, 2005; Kobayashi, 2006). Although some repeats of human rDNA are
organized in palindromic structures that perturb replication fork progression (Caburet et al., 2002; Lebofsky and
Bensimon, 2005), the standard organization model for eukaryotic rDNA consists of clusters of tandem arrays. In
Saccharomyces cerevisiae, one of the major breakthroughs came with the finding that in some if not all of these rDNA
repeats replication forks moving against transcription are blocked at the non-transcribed spacer close to the 3′ end of the
primary transcript (Brewer and Fangman, 1988; Linskens and Huberman, 1988). Identification of replication fork
barriers (RFBs) in the tandem ribosomal repeats of all species studied so far highlights their relevance throughout
evolution (Hernández et al., 1993). It was early on speculated that this replication fork stalling could be due to head-on
collision of transcription and replication (Brewer and Fangman, 1988), but later found that it occurs even in the absence
of transcription (Brewer et al., 1992). The finding that in S. cerevisiae replication fork stalling at the rDNA RFB is
mediated by binding of Fob1p to RFB DNA sequences (Kobayashi and Horiuchi, 1996; Kobayashi and Horiuchi, 1996;
Kobayashi, 2003; Mohanty and Bastia, 2004) ended this debate, although it was recognized that Fob1p is probably not a
lonely player (Krings and Bastia, 2004). Despite replication fork arrest at the rDNA RFBs has been studied profusely,
these are not the only sites where replication forks stall in a programmed manner (reviewed in Hyrien, 2000; Codlin and
Dalgaard, 2003). Notwithstanding, for rDNA RFBs, it is generally accepted that their main role is to prevent head-on
collision between transcription and the replication forks that progress in the opposite direction. These head-on collisions
are thought to have deleterious consequences (Liu et al., 1993; Liu and Alberts, 1995; Elias-Arnanz and Salas, 1997;
1999; Olavarrieta et al., 2002a; Mirkin and Mirkin, 2005; Mirkin et al., 2006). FOB1Δ cells, however, are viable
(Kobayashi and Horiuchi, 1996; Kobayashi, 2003; Mohanty and Bastia, 2004), and in those cells where replication forks
do not stall at the RFBs, there is circumstancial evidence for replication fork stalling due to head-on collision of
transcription and replication (Takeuchi et al., 2003; Krings and Bastia, 2004). Surprisingly, although fork stalling at one
or more ribosomal RFBs occurs in every species studied so far (Hernández et al., 1993), the S. cerevisiae's gene FOB1 is
not conserved and its role in replication fork stalling is taken on by different proteins in different species (Gerber et al.,
1997; López-Estraño et al., 1998; 1999; Sánchez-Gorostiaga et al., 2004; Krings and Bastia, 2004; 2005; Mejía-Ramírez
et al., 2005).
Another interesting debate concerns the relationship between the hyperrecombination hotspot (HOT1) and replication
fork stalling at the RFB (Keil and Roeder, 1984; Kobayashi and Horiuchi, 1996; Kobayashi et al., 1998; 2001). It was
originally thought that the partial coincidence of the RFB and HOT1 indicated they were equivalent, but it was later
shown that HOT1 activity is independent of replication fork arrest (Ward et al., 2000).
A third interesting and related chapter unraveled when it was found that in yeast the histone deacetylase silencing protein
Sir2p regulates recombination at the ribosomal DNA (Gottlieb and Esposito, 1989). It was speculated that SIR2
mutations accelerate ageing through the accumulation of extrachromosomal rDNA circles that form as a consequence of
intramolecular recombination between rDNA repeats (Kaeberlein et al., 1999). Indeed, there is strong evidence
indicating that in S. cerevisiae Sir2p modulates recombination of the replication forks stalled at the rDNA RFBs
(Benguría et al., 2003).
Finally, a very elegant model was recently proposed to explain the homogenization of tandem repeats at the rDNA locus
where cohesion, condensation, non-coding transcription and recombination collaborate to regulate asymmetric
recombination within ribosomal repeats to accomplish successive contractions and expansions of the ribosomal tandem
array (Kobayashi and Ganley, 2005; Kobayashi, 2006).
In the present report we investigated DNA recombination in S. cerevisiae by studying the integration of a circular
minichromosome containing a 35S rDNA minigene and an RFB into specific sites at the chromosomal rDNA locus. The
roles played in this process by replication fork stalling at the ribosomal RFB and due to the collision of transcription and
replication at the 3′ end of the 35S gene were assesed in wild-type, FOB1Δ, SIR2Δ and the double mutant FOB1ΔSIR2Δ
cells.
Results
pBB6RFB+_35SGal (8026 bp) was constructed as described in Experimental procedures (Fig. 1A). It contains the
bidirectional replication origin ARS1, the URA3 gene that allows transformed cells to be selected and the 768 bp
NarI/StuI fragment from the 3′ end of the 35S rDNA unit driven from the GAL10 promoter. Transcription of this 35S
minigene is oriented against the replication fork putatively responsible for its replication. The CYC1 transcription
terminator sequence was placed immediately at the end of the 35S minigene.
Figure 1. Replication fork stalling at specific sites in the circular minichromosome used. A. Map of pBB6RFB+_35SGal
showing the relative position of its most relevant features: the unidirectional ColE1 origin and the gene coding for βlactamase operative in E. coli, and the bidirectional ARS1 replication origin, the URA3 gene and the rDNA minigene
carrying the GAL10 promoter, the 768 bp NarI/StuI rDNA fragment and the transcription terminator CYC1 operative in
S. cerevisiae. The location of recognition sites for a number of restriction endonucleases is shown outside the map. B.
Cartoon of a replication intermediate describing stalling of the counterclockwise (ccw) replication fork at the RFB. C.
Cartoon of a replication intermediate describing stalling of the counterclockwise (ccw) replication fork at the 3′ end of
the minigene due to head-on collision of transcription and replication. Parental strands are depicted in dark blue and
green while nascent strands are depicted in red. RNA transcripts are in light blue.
The rationale for the construction of this minichromosome was as follows: when cells grow in the presence of glucose,
no transcription of the 35S minigene would take place and a counterclockwise progressing replication fork would find no
obstacle to advance through. In those cells grown in the presence of galactose, on the other hand, pol II RNA
polymerases driven from the GAL10 promoter would actively transcribe the 35S minigene and head-on collision
between transcription and counterclockwise progressing replication forks would occur frequently. This would not
necessarily happen in wild-type cells, however, as binding of Fob1p at the RFB would block counterclockwise
progressing replication forks (Fig. 1B). But in FOB1Δ cells these replication forks would find no obstacle to pass
through the RFB and head-on collision between replication and transcription would readily occur provided the 35S
minigene is transcribed (Fig. 1C). As previously mentioned, replication fork stalling is not expected to automatically
trigger DNA recombination as the inaccessible chromatin organization caused by Sir2p would act preventing such DNA
transactions (Benguría et al., 2003 and references therein). In SIR2Δ cells, however, homologous recombination at the
rDNA would be enhanced and integration of the minichromosome at the rDNA chromosomal repeats would be expected
at high rates.
It is important to highlight a significant difference between the minichromosome used here and that one employed in
former studies (Benguría et al., 2003). The presence and orientation of a 35S minigene in pBB6RFB+_35SGal render the
possibility for fork stalling at the 3′ end of the 35S minigene either due to a collision of transcription and replication or at
the CYC1 transcription terminator itself (Fig. 1C). This was not contemplated in former studies, where replication fork
stalling in the minichromosome employed (pBB6-RFB+) could only take place at the RFB.
To identify integration of the minichromosome at the chromosomal rDNA locus we first used agarose gel electrophoresis
of either undigested or restriction digested samples and hybridization with pBR322 DNA used as a probe. pBR322 DNA
hybridizes only to the minichromosome. Linear maps of chromosomal rDNA repeats, with the minichromosome
integrated either at the RFB or at the 3′ end of the 35S gene are depicted in Fig. 2. The recognition sites for a number of
restriction endonucleases are shown. BglII cuts the minichromosome once (leading to an 8.0 kb linear fragment) and
each chromosomal rDNA repeat twice (leading to a 4.4 kb and a 4.6 kb linear fragments, the latter comprising both the 3′
end of the 35S gene and the RFB). After digestion with BglII, integration of the minichromosome at the RFB would lead
to a new reconstituted linear fragment of ∼8.8 kb that results from the addition of ∼6.7 kb (from the minichromosome)
plus ∼2.1 kb (from the chromosomal rDNA repeat). Integration at the 3′ end of the 35S unit would lead to a
reconstituted linear fragment of ∼8.9 kb that results from the addition of ∼6.2 kb (from the minichromosome) plus ∼2.7
kb (from the chromosomal rDNA repeat). In other words, after digestion with BglII and regardless of the integration site,
two linear fragments of 8.0 kb and ∼9.0 kb would be expected, corresponding to unintegrated and integrated copies of
the minichromosome respectively.
Figure 2. Maps of rDNA repeats showing integration of the minichromosome at specific sites by homologous
recombination. A. Integration at the chromosomal RFB. B. Integration at the 3′ end of the 35S chromosomal gene. The
relative location of the most relevant features is indicated for the minichromosome as well as for the chromosomal rDNA
repeat. Vertical bars show the location of recognition sites for a number of restriction endonucleases. The location of
specific primers used for PCR amplification are shown in each case: CYCfwd and 5Srev to check integration at the
chromosomal RFB and CYCrev and 35Sfwd to check integration at the 3′ end of the 35S chromosomal repeat.
Wild-type (CT711) and isogenic FOB1Δ, SIR2Δ and the double-mutant FOB1ΔSIR2Δ cells were transformed with
pBB6RFB+_35SGal and plated in solid agarose media lacking uracil and containing either glucose or galactose plus
raffinose. Cells isolated from colonies 1–2 mm in diameter were patched in new plates containing the same media for
another 4–6 days to expand the cultures. Subsequently, cells corresponding to each clone were seeded in liquid medium
containing, glucose or galactose plus raffinose and grown for 15–20 generations until harvest. In other words, cells were
transformed and plated in media containing glucose or galactose plus raffinose and grown in the same media or switched
to the alternate one for the last 15–20 generations. Finally, DNA was isolated and analysed as described in Experimental
procedures.
Surprisingly, the results obtained were similar regardless of the experimental protocol employed but significantly
different for the different cell strains used. The results obtained with several clones from each strain representing the
different situations observed for out of 15–20 clones analysed in each case are depicted in Figs 3 and 4. For wild-type
cells, undigested samples generated the characteristic bands corresponding to covalently closed circles (CCCs), linears
(Ls) and open circles (OCs). The weak band corresponding to a linear 8.0 kb fragment was likely due to unspecific
double-stranded breakage that occurred during DNA isolation (Fig. 3A, CT711). Restriction digestion of the same DNA
with BglII, on the other hand, generated the single prominent band expected for a linear fragment of 8.0 kb, except for
clone # 1 where a second linear and barely visible band of ∼9.0 kb was observed as well (Fig. 3B, CT711). These results
are in agreement with those obtained before (Benguría et al., 2003) and suggested that almost no integration of the
minichromosome at the rDNA locus occurred in wild-type cells.
Figure 3. Identification of extrachromosomal and integrated copies of pBB6RFB+_35SGal in CT711 wild-type and
FOB1Δ cells. Clones from each strain representing the different situations observed out of 15–20 clones analysed in each
case are shown. Identification in (A) and (B) was by agarose gel electrophoresis, Southern blotting and hybridization
with a labelled probe that only recognizes minichromosome DNA sequences. Undigested and digested samples are
indicated on top of each autoradiogram and the numbers above each lane indicate the clone used. ‘u’ indicates
untransformed cells. Ethidium bromide stained gels at the bottom show the products of PCR amplification for several
clones using the primers indicated in each case. The sizes of the amplified fragments are indicated aside.
Figure 4. Identification of extrachromosomal and integrated copies of pBB6RFB+_35SGal in SIR2Δ and FOB1ΔSIR2Δ
cells. Clones from each strain representing the different situations observed out of 15–20 clones analysed in each case
are shown. Identification in (A–C) was by agarose gel electrophoresis, Southern blotting and hybridization with a
labelled probe that only recognizes minichromosome DNA sequences. Undigested and digested samples are indicated on
top of each autoradiogram and the numbers above each lane indicate the clone used. ‘u’ indicates untransformed cells.
(D) corresponds to identification by agarose gel electrophoresis, Southern blotting and hybridization with a labelled
probe that recognizes minichromosome as well as chromosomal DNA sequences (see Experimental procedures). The
ethidium bromide stained gels at the bottom show the products of a semi-quantitative PCR assay of two clones
normalized to the product obtained with a pair of oligos that amplifies a single copy gene (GPD2), labelled C in each
panel. The size of this amplified fragment was 1011 bp as indicated to the right. Lanes labelled 1 correspond to the
products of PCR amplification using the pair of oligos that revealed integration at the RFB. The size of the amplified
fragment was 1090 bp as indicated to the left. Lanes labelled 2 correspond to the products of PCR amplification using
the pair of oligos that revealed integration at the 3′ end of the 35S gene. In this case the size of the amplified fragment
was 1149 bp as also indicated to the left. Note that in the double mutants FOB1ΔSIR2Δ, integration at the RFB was
significantly less frequent than integration at the 3′ end of the 35S gene.
For FOB1Δ cells the results obtained were similar. In these cells deletion of FOB1 was accomplished by its substitution
with a pBR322 derivative. This explains the detection of a high-molecular-weight smeared signal in undigested samples
(Fig. 3A, FOB1Δ) as well as a 4.9 kb linear band after digestion with BglII (Fig. 3B, FOB1Δ) that actually served as an
internal control. Note that untransformed cells (analysed in the far left lane in each case) generated only those signals
unrelated to the minichromosome. In addition, the undigested samples corresponding to all the clones analysed showed
the typical bands generated by CCCs, Ls and OCs (Fig. 3A, FOB1Δ). Digestion of the samples with BglII converted all
of them into a single band corresponding to a linear fragment of 8.0 kb (Fig. 3B, FOB1Δ). These observations indicated
that as in the case of wild-type cells the minichromosomes remained mostly extrachromosomal in the absence of Fob1p,
too.
The results were totally different when the DNA was isolated from SIR2Δ cells. For undigested samples a distinct OC
band and another very weak band corresponding to CCCs (only visible for the DNA of clone # 1) were detected (Fig.
4A, SIR2Δ). Superimposed on these signals, a high-molecular-weight smeared signal was observed that was prominent
for the DNA of clones # 2 and # 3. In addition, two unexpected bands that could putatively represent CCCs and OCs of a
different minichromosome significantly smaller than the original pBB6RFB+_35SGal were detected for clone # 4. In
agreement with the results obtained for undigested samples, after digestion with BglII, clones # 1, # 2 and # 3 generated
two bands corresponding to linear fragments of 8.0 kb and ∼9.0 kb, corresponding to unintegrated and putatively
integrated forms of the minichromosome respectively. These two bands were clearly detected in 10 out of 17 of the
clones analysed. The relative intensity of the two bands, however, varied significantly among clones. The 8.0 kb band
predominated in clone # 1 whereas the ∼9.0 kb band was more abundant in clone # 2. The intensity of both bands was
alike for clone # 3. Upon digestion with BglII, clone # 4 generated a prominent band corresponding to a linear fragment
of ∼5.4 kb and another barely visible band of ∼6.4 kb (Fig. 4B, SIR2Δ) that could putatively correspond to the
unintegrated and integrated forms of the new small version of the minichromosome respectively.
To confirm these results we digested the same samples with a different restriction enzyme. NsiI cuts
pBB6RFB+_35SGal only once close to the 3′ end of the URA3 gene (Fig. 1A). There is also a single NsiI site in each
rDNA repeat (Fig. 2). Therefore, after restriction digestion with this enzyme unintegrated minichromosomes were
expected to generate a single band corresponding to a linear fragment of 8.0 kb. Integration of the minichromosome at
the chromosomal rDNA locus would lead to a linear fragment of ∼11.3 kb that results from the addition of ∼5.0 kb
(from the minichromosome) plus ∼6.3 kb (from the chromosomal rDNA repeat). These two bands were clearly visible in
the lanes corresponding to clones # 1, # 2 and # 3 (Fig. 4C, SIR2Δ). As occurred after digestion with BglII, digestion of
the DNA from clone # 4 with NsiI generated a single band corresponding to a linear fragment of ∼5.4 kb. To further
confirm these results we digested the DNA isolated from these clones with BglII but now hybridized the membrane with
an rDNA probe (the 768 bp NarI/StuI fragment of the 35S gene). The results are shown in Fig. 4D (SIR2Δ). DNA
isolated from untransformed cells was analysed in the far left lane. As the probe recognized DNA sequences from the
minichromosomes as well as from the chromosomal rDNA locus, in the absence of integration two bands should be
detected corresponding to fragments of 8.0 kb (the linearized minichromosome) and 4.6 kb (one of the two fragments
that results after digestion of each chromosomal rDNA repeat with BglII). The latter was the only band detected in the
DNA isolated from untransformed cells (Fig. 4D, left lane, SIR2Δ). The same band was detected also in the DNA
isolated from all the transformed clones. In addition, the DNA isolated from clones # 1, # 2 and # 3 generated two new
distinct bands corresponding to linear fragments of ∼9.0 kb and ∼3.4 kb respectively (Fig. 4D, SIR2Δ). These are
precisely the bands expected if the minichromosome integrates at the chromosomal rDNA. No such bands were detected
for the DNA of clone # 4. Altogether these results indicated that in SIR2Δ cells the minichromosome integrated at the
rDNA locus quite frequently, although to different extents in the cells corresponding to different clones. The
minichromosomes isolated from clone # 4 seemed to have experienced a rearrangement such that its size was reduced
from 8.0 kb to approximately 5.4 kb (see below).
Contrary to the results obtained in former studies for pBB6-RFB+ (Benguría et al., 2003), where almost no integration
occurs in FOB1ΔSIR2Δ cells, integration of pBB6RFB+_35Gal was evident in the double mutants in this case. Although
not identical, the results obtained when the DNA was isolated from these double mutants were similar to those obtained
with DNA isolated from SIR2Δ cells with just one outstanding exception. For undigested samples hybridized with
pBR322 DNA as a probe, superimposed on the barely visible signals corresponding to CCCs and OCs, a high-molecularweight smeared signal was clearly observed for the DNAs of clones # 1 and # 2. On the contrary, the DNA isolated from
clone # 3 generated almost exclusively the signals corresponding to CCCs, Ls and OCs. After digestion with BglII, the
DNA isolated from clone # 1 generated three prominent bands corresponding to linear fragments of 4.9, 8.0 and ∼9.0 kb
respectively. The 8.0 kb band was absent in the DNA corresponding to clone # 2 and the ∼9.0 kb band was missing in
the lane corresponding to clone # 3 (Fig. 4B, FOB1ΔSIR2Δ). In these cells, the 9.0 kb band that served as a reporter for
minichromosome integration, was observed in six out of nine of the clones analysed. It should be noted, though, that as
explained before, deletion of FOB1 was accomplished by its substitution with a pBR322 derivative. This explains the
detection of a high-molecular-weight smeared signal in undigested samples as well as a linear band of 4.9 kb after
digestion with BglII (Fig. 4A and B, FOB1ΔSIR2Δ). Note that untransformed cells (analysed in the far left lane in each
case) generated only those signals unrelated to the minichromosome.
Altogether, these results indicated that for the double mutant cell line FOB1ΔSIR2Δ, extrachromosomal as well as
integrated copies of the minichromosomes coexisted for clone # 1. Almost all minichromosomes had integrated at the
rDNA locus for clone # 2 although no Fob1p was present in these cells, and no integration was detected for clone # 3.
These results were confirmed by digesting the DNA with NsiI (Fig. 4C, FOB1ΔSIR2Δ) and hybridization with an rDNA
probe after digestion with BglII (Fig. 4D, FOB1ΔSIR2Δ).
The results obtained so far indicated that for pBB6RFB+_35SGal in SIR2Δ cells, integration of the minichromosome at
the chromosomal rDNA locus occurred quite frequently, regardless of Fob1p. These results, however, did not allow us to
determine whether such integration occurred at the RFB or at the 35S gene.
To determine the precise site of integration we designed specific oligos that were complementary to either the
minichromosome or the chromosomal rDNA repeat alone. Appropriate pairs of these oligos, where one of them
hybridized only to the minichromosome and the other one to the chromosomal rDNA repeat, were used to amplify by
PCR the DNA of each of the clones analysed. These oligos are depicted in Fig. 2A and B. To identify integration at the
RFB of the chromosomal rDNA the pair of oligos used was CYCfwd (which has complementary sequences only to the
minichromosome) and 5Srev (which has no complementary sequences to the minichromosome and hybridizes only to
the chromosomal rDNA). To identify integration of pBB6RFB+_35SGal at the 3′ end of the 35S gene in the
chromosomal rDNA, the pair of oligos used was 35Sfwd (which hybridizes only to the chromosomal rDNA) and
CYCrev (which has no complementary sequences to the chromosomal rDNA and hybridizes only to the
minichromosome). In this way, we were certain that PCR amplification could only occur if there had been integration of
the minichromosome into chromosomal rDNA repeats.
The DNA sequence that should be putatively amplified in both cases is shown in Fig. 5. To our surprise, almost all the
clones analysed were positively amplified by PCR using the oligos described above with only two exceptions. Moreover,
the sizes of the amplified bands were those expected for integration of the minichromosome at the RFB as well as at the
3′ end of the 35S gene (1090 and 1149 bp respectively). The results obtained are shown below the autoradiograms
corresponding to each cell line in Figs 3 and 4. To double check these unexpected results, the amplified bands were
sequenced and in all cases the results confirmed integration at the site determined by the pair of oligos that had been
used. Due to the large size of the amplicon, no real-time PCR was possible. This notwithstanding, a semi-quantitative
assay normalized to the product obtained with a pair of oligos that allows amplification of a single copy gene (GPD2),
revealed that while the integration frequency at both sites was equivalent for SIR2Δ cells, in the double mutants
(FOB1ΔSIR2Δ) it occurred significantly more often at the 3′ end of the 35S gene than at the RFB (see the photographs
of ethidium bromide stained gels shown at the bottom of Fig. 4, SIR2Δ and FOB1ΔSIR2Δ).
Figure 5. Details of the DNA sequences amplified by PCR after integration of the minichromosome at the chromosomal
RFB (on top) and at the 3′ end of the 35S chromosomal repeat (at the bottom). On top, the primers employed are
indicated in magenta. Following the CYCfwd primer, the sequence in black corresponds to the CYC1 transcription
terminator that is only present in the minichromosome. The sequence in red corresponds to the RFB that is present in
both the minichromosome and the chromosomal rDNA repeats. The sequence in black between the RFB and the 5Srev
primer corresponds to the 5S that is only present in the chromosomal rDNA repeats. At the bottom, the primers
employed are indicated in brown. Following the 35Sfwd primer, the sequence in light blue corresponds to the 35S gene
that is only present in the chromosomal repeats. The sequence in dark blue corresponds to the 768 bp NarI/StuI fragment
that is present in both the minichromosome and the chromosomal rDNA repeats. The sequence in magenta between the
768 bp NarI/StuI fragment and the CYCrev primer (highlighted in brown) corresponds to the CYC1 transcription
terminator that is only present in the minichromosome.
No PCR amplification was observed for two of the clones analysed: clone # 4 in SIR2Δ cells and clone # 2 in the double
mutant FOB1ΔSIR2Δ cells (data not shown). These results served as negative controls as well. For clone # 4 in SIR2Δ
cells a deletion was suspected as in this case the linear minichromosome was 5.4 kb as compared with the 8.0 kb of the
full pBB6RFB+_35SGal. The deletion could only have affected the region between the URA3 gene and the replication
origin ARS1 as the cells grew normally in a media lacking uracil (see Fig. 1). To confirm this assumption we used PCR
to attempt DNA amplification of the new minichromosome of this clone using a new pair of oligos: URAfwd and
ARS1rev (see the map in Fig. 6A). An amplified linear fragment of 1219 bp was clearly detected (Fig. 6A). DNA
sequencing revealed that this fragment lacked the GAL10 promoter, the 768 pb NarI/StuI 35S DNA sequence as well as
the CYC1 transcription terminator but kept the RFB (data not shown). Moreover, assessment of the regions adjacent to
the deletion revealed the presence in pBB6RFB+_35SGal of two identical 35-bp-long DNA sequences from pBR322 that
were carried on during construction of the minichromosome (Fig. 6A). We speculated that homologous recombination
caused the intervening DNA to pop out leading to a new stable minichromosome of 5.4 kb.
Figure 6. Analysis of the DNA isolated from those clones that failed to amplify by PCR using the primers described
before. A. Map of pBB6RFB+_35SGal showing the relative position of its most relevant features. The identical DNA
sequence of the two fragments flanking the rDNA minigene (highlighted in red) is indicated. The ethidium bromide
stained gel to the right corresponds to the product of PCR amplification using the primers indicated in the map. Both
primers used hybridize only to the minichromosome. B. Integration at the chromosomal RFB of the minichromosome of
clone #4 of SIR2Δ cells lacking the rDNA minigene. The relative location of the most relevant features is indicated for
the modified minichromosome and for a chromosomal rDNA repeat. Vertical bars show the location of recognition sites
for a number of restriction endonucleases. The location of ARS1rev and URAfwd primers (that hibridize only to the
minichromosome) as well as 35Sfwd and 5Srev (that hibridize only to the chromosomal repeats) used for PCR
amplification is shown. At the bottom, ethidium bromide stained gels show the products of PCR amplification for clone
# 4 from SIR2Δ cells and clone # 2 from the double mutant FOB1ΔSIR2Δ cells using the primers indicated above each
gel. In all cases, the size of the amplified fragments is indicated aside.
To check for integration of this new minichromosome at the chromosomal rDNA, new pairs of oligos were used. For the
first pair, URAfwd (which has complementary sequences only to the minichromosome) replaced CYCfwd while 5Srev
(which has no complementary sequences to the minichromosome and hybridizes only to the chromosomal rDNA) was
maintained. For the second pair of oligos, 35Sfwd (which hybridizes only to the chromosomal rDNA) was maintained
and ARS1rev (which has no complementary sequences to the chromosomal rDNA and hybridizes only to the
minichromosome) was used to replace CYCrev (see the linear map in Fig. 6B). The results obtained revealed that this
smaller version of the minichromosome integrated into the rDNA (Fig. 6B, clone # 4 SIR2Δ). DNA sequencing of the
band amplified with URAfwd and 5Srev (2118 bp long) confirmed that integration occurred exclusively at the RFB (data
not shown).
A PCR amplification product was also not observed for the DNA isolated from clone # 2 in the double mutant
FOB1ΔSIR2Δ. To identify the cause for this failure, PCR amplification was attempted using the new pairs of oligos
described above. PCR amplification with the new pairs of oligos was successful again (Fig. 6B, clone # 2,
FOB1ΔSIR2Δ). DNA sequencing of the amplified band revealed that in this case too integration occurred exclusively at
the RFB (data not shown). DNA sequencing allowed us to identify the cause for this unexpected result as well. The
amplified band from clone # 2 of the double mutants lacked the DNA sequences corresponding to the CYC1
transcription terminator. This observation explains why PCR amplification using CYCfwd and CYCrev oligos have
failed.
Discussion
As more and more information on the organization of the genomes of different species unravels, the idea that redundancy
developed and is maintained because it provides genetic advantage, steadily gains support (Thomas, 1993; Cooke et al.,
1997). This applies to polar terminators of DNA replication in Escherichia coli and Bacillus subtilis (Bussiere and
Bastia, 1999; Neylon et al., 2005; Mulcair et al., 2006), cdc-like kinases that phosphorylate specific serine/arginine (SR)
proteins that modulate premRNA splicing in vertebrates (García-Sacristán et al., 2005) as well as cyclin-dependent
kinases that are required to drive cells through interphase in mammals (Santamaría et al., 2007). Maintenance of
homogeneity within repeated families of DNAs appears to be no exception. It was early recognized that this
homogenization occurs via homologous DNA recombination (Smith, 1976; Coen et al., 1982; Nagylaki and Petes, 1982;
Hillis et al., 1991; Schlotterer and Tautz, 1994). Several models were proposed to explain the mechanisms that promote
homologous recombination at specific DNA loci. Among them, replication fork stalling is often preferred as it provides
free DNA ends that are known to be highly recombinogenic (Smithies et al., 1985; Prado and Aguilera, 2005). The
finding of RFBs at the rDNA repeats (Brewer and Fangman, 1988; Linskens and Huberman, 1988) and the observation
that these rDNA RFBs occur in all organisms studied so far (Hernández et al., 1993; Little et al., 1993; Wiesendanger et
al., 1994; Zhang et al., 1997; López-Estraño et al., 1997; 1998; Sánchez-Gorostiaga et al., 2004) strengthen this idea.
Finding that some of the proteins involved in replication fork stalling at these RFBs are not conserved and, furthermore,
the presence of several polar RFBs in each repeat (Kobayashi and Horiuchi, 1996; Gerber et al., 1997; López-Estraño et
al., 1998; Ward et al., 2000; Putter and Grummt, 2002; Kobayashi, 2003; Mohanty and Bastia, 2004; SánchezGorostiaga et al., 2004; Mejía-Ramírez et al., 2005) indicate that the efficiency of these elements to stall replication forks
is rather poor. In other words, some replication forks only pause and are able to pass through thereafter or the proteins
involved are not always there. One of the first hypotheses formulated to justify the presence of these rDNA RFBs was
that their main role would be to prevent head-on collision of transcription and replication (Brewer and Fangman, 1988;
Linskens and Huberman, 1988; Kobayashi, 2003). It is also possible, though, that in addition another role would be to
promote homologous recombination.
As expected from previous studies (Benguría et al., 2003), the results obtained for pBB6RFB+_35SGal confirmed that
integration of the minichromosome at the chromosomal rDNA RFB locus was significantly enhanced in SIR2Δ cells
(Fig. 4A–D, SIR2Δ). Contrary to the results obtained for pBB6-RFB+ (Benguría et al., 2003), however, here we showed
that pBB6RFB+_35SGal integrated efficiently at the rDNA locus regardless of Fob1p. This apparent contradiction is
explained by the presence and orientation of a 35S minigene in pBB6RFB+_35SGal. There is no such minigene in
pBB6-RFB+ (Benguría et al., 2003). Moreover, the semi-quantitative PCR assay performed here indicated that in the
double mutants integration occurred more often at the 3′ end of the 35S gene. This observation indicated that in
FOB1ΔSIR2Δ cells homologous recombination was preferentially triggered by fork stalling at the 3′ end of the 35S gene.
PCR amplification and DNA sequencing revealed also that integration occurred at both sites, albeit significantly less
frequently, in wild-type cells (Fig. 3C and D, CT711) and even in the absence of Fob1p (Fig. 3C and D, FOB1Δ). These
observations confirmed that homologous recombination at the rDNA RFB is promoted by more than one mechanism. As
previously mentioned, it was originally thought that the hyperrecombination site HOT1 (Keil and Roeder, 1984) was
linked to replication fork stalling at the RFB (Kobayashi and Horiuchi, 1996), but it was later shown that HOT1 activity
is independent of replication fork arrest at the RFB as evidenced by two-dimensional agarose gel electrophoresis (Ward
et al., 2000). Our results fully agree with the latter observation, and it is tempting to speculate that HOT1 activity could
be more closely related to DNA topoisomerase I cleavage at several specific and conserved sites within this region
(Ganley et al., 2005). This cleavage is independent of replication and transcription (Burkhalter and Sogo, 2004; DiFelice
et al., 2005). Although it is speculated that binding to DNA of Fob1p might represent by itself a signal for this DNA
topoisomerase I activity, some cleavage was observed even in FOB1Δ cells, suggesting that binding of Fob1p enhances
topoisomerase I cleavage but it is not absolutely required. Therefore, this activity could be linked to HOT1 and would
explain the integration of pBB6RFB+_35SGal we have found at the chromosomal rDNA RFB locus in FOB1Δ cells
(Fig. 3C, FOB1Δ).
In those cases where fork stalling at the RFB was avoided, collision of transcription and replication could occur. We
attempted to control transcription from the GAL10 promoter in the minichromosome by growing the cells in the
presence of glucose or galactose. Glucose inhibits transcription driven from the GAL10 promoter. The transcriptional
regulation of GAL genes, however, is complex involving dynamic chromatin transitions that continue to fluctuate after
switching from glucose to galactose and vice-versa (Cavalli and Thoma, 1993; Lohr et al., 1995). We speculate this
might explain why repression of the GAL10-driven 35S minigene in the multicopy minichromosome was leaky in the
presence of glucose, allowing some transcription to take place. This residual transcription could have been responsible
for stalling replication forks moving in the opposite direction upon head-on collision at the 3′ end of the 35S minigene
when the cells were grown in the presence of glucose.
An alternative explanation is that the CYC1 transcription terminator acts as an RFB by itself. In the literature there are
several examples of DNA elements that serve dual purposes. Some of these elements terminate transcription on one side
and stall replication forks progressing in the opposite direction on the other. Among them, the rDNA transcription
terminators mTTF-I bound to Sal boxes in the mouse (Grummt et al., 1985; Gerber et al., 1997; López-Estraño et al.,
1998) as well as RFB2 and RFB3 in Schizosaccharomyces pombe (Krings and Bastia, 2004; Sánchez-Gorostiaga et al.,
2004). CYC1 contains a dyad symmetry element that can easily form a stem and loop structure (Zaret and Sherman,
1982). This type of structures are known to pause or even stall replication forks (Mirkin and Mirkin, 2007). In short, in
some cells replication fork stalling at the 3′ end of the 35S gene could have occurred at the CYC1 transcription
terminator in the minichromosome and due to collision of transcription and replication machineries in the chromosome.
This would explain those unexpected cases of minichromosome integration at the 3′ end of the chromosomal 35S gene
(Fig. 3, CT711 35Sfwd/CYCrev, FOB1Δ 35Sfwd/CYCrev, and Fig. 4, SIR2Δ, lane 2 of clones # 5 and 7, and
FOB1ΔSIR2Δ, lane 2 of clones # 1 and 3 respectively). This latter interpretation was strengthened after finding that no
integration at this site occurred when the minichromosome lacked the CYC1 transcription terminator, as in clone # 2 of
the double mutant FOB1ΔSIR2Δ (Fig. 6, clone # 2, FOB1ΔSIR2Δ).
Experimental evidence for replication fork stalling due to the collision of transcription and replication is not abundant in
the literature. In vitro assays show that such collisions lead to a short pausing of the replication forks (Liu et al., 1993;
Liu and Alberts, 1995; Elias-Arnanz and Salas, 1997; 1999). On the other hand, direct evidence for replication fork
stalling in vivo is documented using two-dimensional agarose gel electrophoresis for just a few cases: a bacterial plasmid
carrying an inducible promoter (Mirkin and Mirkin, 2005), tRNA genes in S. cerevisiae (Deshpande and Newlon, 1996),
rDNA in S. pombe (Krings and Bastia, 2004) and a FOB1Δ strain of S. cerevisiae with just ∼20 rDNA repeats (Takeuchi
et al., 2003). The observation that in the rDNA of wild-type S. pombe cells this collision generates a discrete signal in
two-dimensional gels (Krings and Bastia, 2004) indicates that the probability for such collisions to occur at the 3′ end of
the 35S gene is the highest. This observation fits well with the notion that each actively transcribing 35S gene is literally
burden with RNA polymerase I molecules to generate the classical ‘christmas trees’ observed at the electron microscope
(Miller and Beatty, 1969). Data suggesting that replication fork stalling due to the collision of transcription and
replication stimulates DNA recombination was only obtained for the low containing rDNA repeats strain of S. cerevisiae
(Takeuchi et al., 2003) and outside the rDNA locus (Prado and Aguilera, 2005).
From a quantitative viewpoint, the results obtained indicate that the absence of Sir2p notably enhanced integration of the
minichromosome at the rDNA locus. This is in agreement with the notion that Sir2p creates a closed chromatin structure
at this locus that affects gene silencing and recombination (Gottlieb and Esposito, 1989; Fritze et al., 1997; Gasser and
Cockell, 2001; Benguría et al., 2003). As previously mentioned, though, the minichromosome's integration reporter band
did not vary significantly between SIR2Δ and the double mutant FOB1ΔSIR2Δ cells (Fig. 4). This observation indicates
that homologous recombination at the rDNA is not necessarily dependent on replication fork stalling at the RFB
mediated by Fob1p.
It is important to realize the natural dynamics of the rDNA locus throughout consecutive generations. As previously
mentioned, contraction and expansion of this locus are constant and responsible for the homogeneization of rDNA
repeats (Smith, 1976; Coen et al., 1982; Nagylaki and Petes, 1982; Hillis et al., 1991; Schlotterer and Tautz, 1994). In
other words, as opposed to unique DNA sequences in the genome, rDNA tandem repeats are highly unstable. For this
reason the significance of performing a quantitative PCR assay to determine the frequency of minichromosome
integration in each case was uncertain as even in a population of cells derived from a single clone, the number of
integrated minichromosomes is expected to vary from cell to cell. Moreover, it is well known that the presence of
extrachromosomal DNA, whatever its nature, represents an energetic cost that decreases longevity of these cells as
opposed to those lacking episomes (Falcon and Aris, 2003). On the other hand, Sir2p is also involved in silencing the
expression of foreign genes transcribed by RNA polymerase II when integrated at the rDNA locus (Smith and Boeke,
1997). Altogether, these observations highlight the fact that opposing factors affecting the stability of integrated copies
of the minichromosome at the rDNA locus were continuously at work in the different cell types used in this study.
The role of replication fork stalling at the RFB in those processes where contraction and expansion of rDNA were
observed still remains controversial. It was early shown that the absence of an essential subunit of RNA polymerase I in
rpa135 deletion mutants triggers a gradual contraction of rDNA repeats. Reintroduction of the missing gene induces an
expansion of repeat numbers back to normal. Using this system it was found that FOB1 is essential for both the
contraction and expansion of rDNA repeats and it was suggested that both events are triggered by FOB1-dependent
pausing of replication forks at RFB sites (Kobayashi et al., 1998). It was also found that strains defective in the
transcription factor UAF give rise to variants that are able to transcribe rDNA genes by RNA polymerase II. In these
variants there is a concomitant FOB1-dependent expansion of chromosomal rDNA repeats to levels severalfold higher
than in wild-type cells (Oakes et al., 1999). More recently, Grenetier et al. (2006) reported that in CTD kinase I null
mutant strains there is a significant reduction in the number of tandem rDNA repeats. Reintroduction of the missing gene
induces an expansion of rDNA repeats to reach a copy number similar to that of wild-type strains. Surprisingly though,
in this system while expansion is dependent of Fob1p, contraction still occurs in its absence (Grenetier et al., 2006).
It seems obvious that both contraction and expansion occur via homologous DNA recombination. This requires
replication fork stalling and fork stalling at RFBs is significantly enhanced in FOB1 cells. As clearly demonstrated here
and elsewhere though (Takeuchi et al., 2003; Krings and Bastia, 2004), fork stalling and homologous recombination at
rDNA repeats still takes place in the absence of Fob1p. It would be very interesting to know if fork stalling at the 3′ end
of the 35S gene due to the collision of transcription and replication predominates in those cases where contraction was
found to be FOB1 independent. In this context it is important to note that Fob1p has more than a single function, as it
was recently shown that it also plays a significant role in rDNA disjunction during anaphase (Machin et al., 2006).
Finally, transcription and initiation of DNA replication at the chromosomal rDNA locus are clustered (Muller et al.,
2000; Pasero et al., 2002). This makes certain that contraction and expansion, regardless of whether recombination
occurs at the RFB or at the 3′ end of the 35S gene, take place stepwise.
In summary, here we showed that integration of the minichromosome at the rDNA locus occurred only at two distinct
sites, the RFB and the 3′ end of the 35S gene. These integration events were subjected to replication fork stalling at these
sites in both the minichromosome and the chromosomal repeats. Fork stalling at the 3′ end of the 35S gene in the
chromosome is a consequence of head-on collision of transcription and replication. Stalling at the RFB, on the other
hand, is enhanced by the presence of Fob1p but it also occurs without it. Based on these results we propose that one of
the main roles for replication fork stalling at the rDNA locus could be to promote homologous recombination rather than
just to prevent head-on collision of transcription and replication as originally thought. We speculate this potential allows
cells to homogenize and either contract or expand rDNA to adapt to different situations.
Experimental procedures
Yeast strains and culture medium
The CT711 yeast strain (MATa, leu2–3, 113, his3Δ1, trp1, ura3–52, ade2–101, can1) and the three isogenic mutants
used are described elsewhere (Benguría et al., 2003). Cells were transformed using the acetate lithium method described
in the Current Protocols in Molecular Biology (Ausbubel et al., 1995) and were grown at 30°C in synthetic medium
without uracil, containing 2% glucose or 2% galactose plus 2% raffinose (SC-uracil). To make solid media 2% agar was
added.
Plasmid construction
pBB6RFB+_35SGal (8026 bp) was constructed in three steps. In the first step, the 1683 bp StuI 35S-rDNA fragment of
pRIB-S1 (constructed and kindly provided by J. Warner, Albert Einstein College of Medicine) was inserted at the unique
SmaI site of pUC19. In the second step, the 784 bp NarI/SalI fragment from the resulting plasmid (containing the 768 bp
NarI/StuI 35S-rDNA fragment) was inserted between the ClaI/SalI sites of p306GALCYC containing the GAL10
promoter and the CYC1 transcription terminator. In the third and final step, the 3540 pb NdeI/KpnI fragment containing
the URA3 gene, the GAL10 promoter, the 768 pb NarI/StuI 35S fragment and the CYC1 transcription terminator was
inserted between the NdeI/NsiI sites of pBB6-RFB+@BstXI (constructed and kindly provided by Bonita Brewer,
University of Washington) containing the ARS1 origin and the RFB.
Yeast DNA isolation
Yeast DNA used for plasmid integration analysis was obtained from 10 to 20 ml of saturated yeast cultures (1.5 × 108
cell ml−1) after the cells grew for ∼36 generations in plates or ∼20 generations in liquid medium (Takeuchi et al.,
2003). DNA was obtained by breaking the cells with acid-washed glass beads in 200 μl lysis buffer (10 mM Tris, pH 8.0,
1 mM EDTA, 100 mM NaCl, 1% SDS, 2% Triton X-100) plus 200 μl PCIA (25:24:1 phenol:chloroform:isoamyl
alcohol). After vortex during 2 min at 4°C, TEN100 was added (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl). The
aqueous phase was recovered and this step was repeated once. Finally, the aqueous phase was extracted three times with
PCIA and once with CIA before the DNA was precipitated with 1 ml cold ethanol. The pellet was dried and dissolved in
30 μl H2O. The DNA isolated from 2.5 to 5.0 × 109 cells was used for Southern blot analysis of intact or digested DNA
molecules respectively. DNA was electrophoresed in 0.7% agarose in 1× TBE buffer and subjected to Southern
hybridization using pBR322 as a probe. pBR322 hybridizes only to the minichromosome and not to chromosomal rDNA.
Southern hybridization
Agarose gels were transferred to nylon membranes (Zeta-Probe; Bio-Rad) and hybridized with probes labelled by the
Random Primer Fluorescein Labeling Kit (NENTM.; Life science Products) as described before (Olavarrieta et al.,
2002b; Benguría et al., 2003).
PCR analysis
PCR was used to confirm integration of the minichromosome into chromosomal rDNA repeats. CYCfwd (which only
recognizes minichromosome DNA) and 5Srev (which only recognizes chromosomal DNA) were used to confirm
integration of the minichromosome at the RFB of chromosomal rDNA. 35Sfwd (which only recognizes chromosomal
DNA) and CYCrev (which only recognizes minichromosome DNA) were used to confirm integration of the
minichromosome at the chromosomal 3′ end of the 35S gene. The single-copy gene coding for glyceraldehyde-3phosphate dehydrogenase (GPD2) was amplified as an internal control. In other experiments, URAfwd and ARS1rev
were used to replace CYCfwd and CYCrev respectively. Total genomic DNA was extracted from 109 cells. PCR was
performed with RighTaq polymerase (EuroClone) in 30 cycles with the following profile for each cycle : denaturating at
95°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 2 min. The PCR products were electrophoresed in
0.8% agarose gels and stained with EtBr. Densitometry was performed using NIH Image 1.6 normalizing with the GPD2
controls where indicated.
The DNA sequence of the primers used was as follows:
CYCfwd: 5′-GCTTGAGAAGGTTTTGGGACGCT-3′
5Srev: 5′-TCCCATAACTAACCTACCACTCG-3′
35Sfwd: 5′-CATTGTGAAGAGACATAGAGGGTG-3′
CYCrev: 5′-AGCGTCCCAAAACCTTCTCAAGC-3′
URAfwd: 5′-CTAAGGTAGAGGGTGAACGTTAC-3′
ARS1rev: 5′-CTGCACTGAGTAGTATGTTGCAG-3′
GPD2fwd: 5′-CTTAAGCGAACGCATCCGGTGTT-3′
GPD2rev: 5′-CTGCAACACCAGCGGATTCTTGA-3′
Acknowledgements
We acknowledge María Pilar Robles for technical assistance and Alberto Benguría, Marta Fierro-Fernández, María José
Fernández-Nestosa, Leonor Rodríguez, Eva Mejía, Raúl Torres, María Tenorio and Zaira García for their suggestions
and support during the course of this study. This work was sustained in part by Grants No. BIO2005-02224 to J.B.S. and
BFU2007-62670 to P.H. from the Spanish Ministerio de Educación y Ciencia.
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