Identification of DNA cis elements essential for expansion of ribosomal DNA repeats in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae carries approximately 150 ribosomal DNA (rDNA) copies in tandem repeats. Each repeat consists of the 35S rRNA gene, the NTS1 spacer, the 5S rRNA gene, and the NTS2 spacer. The FOB1 gene was previously shown to be required for replication fork block (RFB) activity at the RFB site in NTS1, for recombination hot spot (HOT1) activity, and for rDNA repeat expansion and contraction. We have constructed a strain in which the majority of rDNA repeats are deleted, leaving two copies of rDNA covering the 5S-NTS2-35S region and a single intact NTS1, and whose growth is supported by a helper plasmid carrying, in addition to the 5S rRNA gene, the 35S rRNA coding region fused to the GAL7 promoter. This strain carries a fob1 mutation, and an extensive expansion of chromosomal rDNA repeats was demonstrated by introducing the missing FOB1 gene by transformation. Mutational analysis using this system showed that not only the RFB site but also the adjacent approximately 400-bp region in NTS1 (together called the EXP region) are required for the FOB1-dependent repeat expansion. This approximately 400-bp DNA element is not required for the RFB activity or the HOT1 activity and therefore defines a function unique to rDNA repeat expansion (and presumably contraction) separate from HOT1 and RFB activities.

FIG. 1

(A) Structure of rDNA repeats in S. cerevisiae. The locations of the 35S and 5S rRNA genes (with the direction of transcription indicated by arrows), the two nontranscribed spacer regions (NTS1 and NTS2), ARS (replication origin), and the HOT1 I-element are shown in the upper part. BglII A and B DNA fragments are also shown. NTS1 and its surrounding regions are expanded. Three solid bars represent the HOT1 E-element, Pol I Enhancer, and RFB (the replication fork blocking site, also indicated by ). (B) Structure of pRDN-hyg1 (4). This plasmid carries a single copy of rDNA repeats obtained by cutting the repeats with SmaI (hence the copy starting from −206 and ending at −207; the numbering is with respect to the Pol I transcription start site as +1). There is a mutation in the 18S rRNA gene (indicated as an asterisk) which makes the ribosome hygromycin B resistant. (C) Structure of pNOY353. This plasmid carries the 7.5-kb BamHI-XhoI fragment, which contains GAL7-35S rDNA (the 35S rRNA coding region fused to the GAL7 promoter as described by Nogi et al. [23]) inserted between the BamHI and SalI sites of the pTV3 vector (27). This plasmid also contains the 1,085-bp PvuII-EcoRV fragment carrying the 5S rRNA gene (see panel A) inserted in the SmaI site upstream of the GAL7 promoter. The 35S rRNA coding region contains up to the HindIII site, +6935. Thus, the Pol I enhancer is present but the region from HindIII to PvuII in NTS1 and the Pol I promoter region (from −1 to the EcoRV site at +8757 or the 381-bp region) are absent.

FIG. 2

The fork block-dependent recombination model for rDNA repeat expansion and contraction. The positions of ARS and RFB are shown as solid dots and , respectively. Individual lines represent chromatids with double-stranded DNA. In this model, DNA replication starts from one of the ARS sites (ARS-2) bidirectionally (a). In the yeast rDNA repeats, about one in five ARS sites is used as an active origin (2, 21). A rightward replication fork is arrested at the RFB site, and this arrest is supposed to stimulate a double-strand break of DNA at a nearby site (indicated by an arrowhead in row b). A strand invasion at a homologous duplex (a downstream sister chromatid near ARS-1 in this example) takes place (c), and a new replication fork is formed. The new replication fork meets with the leftward replication fork from the upstream site, resulting in formation of two sister chromatids, one of which gains an extra copy of rDNA, indicated as boxed rDNA-2 (d). If the strand invasion is at a site in a upstream repeat (e.g., near ARS-3), a loss, rather than a gain, of an rDNA repeat is expected. This model was proposed previously to explain the observed strong dependence of rDNA repeat expansion and contraction on FOB1 (18).

FIG. 3

(A) Analysis of the size of chromosome XII by CHEF electrophoresis. Eight independent hygromycin-resistant mutants (lanes 1 to 8), as well as the control strain, NOY408-1b, (lane WT), were examined. The left panel shows chromosome patterns revealed by staining with EtBr. The right panel shows an autoradiogram obtained after hybridization with an rDNA probe (probe 3 in Fig. 5C). Size markers (lane M) are made up of Hansenula wingei chromosomes (Bio-Rad). (B) Analysis of the sizes of rDNA repeats by field inversion gel electrophoresis. DNA samples prepared from mutants 7 and 8 (those shown in lanes 7 and 8 in panel A, respectively) were digested with BamHI, subjected to the electrophoresis, and analyzed by hybridization using an rDNA probe (probe 3 in Fig. 5C). A band seen in lane 8 which corresponds to the size expected from two copies of rDNA is indicated by an arrowhead. Lane M is the 5-kb ladder provided by Bio-Rad. Because the amounts of the marker DNA molecules were much larger than the amount of fragment containing two copies of rDNA, nonspecific hybridization of the probe to the markers took place, providing positions of the markers conveniently on the same autoradiogram. (C) Structures of two rDNA repeats remaining in strain TAK201 and the NTS1-5S region subjected to the mutational analysis (expanded below). Seven segments, A to G, replaced by URA3 individually in mutants A to G are indicated. The precise positions of each segment are given in Materials and Methods.

FIG. 4

Analysis of the size of chromosome XII in FOB1 transformants of NTS1 substitution mutants by CHEF electrophoresis. (A and B) Five independent FOB1 transformants derived from each of mutants A to G and from the control strain (TAK201) were analyzed along with five vector transformants of the control strain after ∼45 generations. The reference TAK201, which had two rDNA repeats without expansion, was also analyzed (lane 2-copies). (A) Chromosomal patterns revealed by staining with EtBr; (B) autoradiograms obtained after hybridization with an rRNA probe (probe 3 in Fig. 5C). (C) Analysis of five FOB1 transformants and five vector transformants derived from mutant G by hybridization using a URA3 probe. The left panel shows chromosomal patterns revealed by staining with EtBr. The right panel shows an autoradiogram obtained after hybridization with the URA3 probe. The position of chromosome V carrying the native URA3 gene is indicated by an asterisk. On the right sides of the gels in panels A and B and on the left side in panel C, the positions of chromosomes and their sizes (in megabases) are indicated.

FIG. 5

Expansion of rDNA repeats observed in FOB1 transformants of NTS1 substitution mutants. (A) The DNA samples analyzed in the experiments in Fig. 4A and B were digested with BglII and analyzed by Southern hybridization using rDNA-specific probes, probe 2 for the left panel and probe 1 for the right panel (the probes are indicated in panel C). The gels were also analyzed using a probe specific for a single-copy gene, MCM2, as a reference. (B) The numbers of rDNA repeats was calculated for each transformant, and the values for five independent transformants derived from each mutant and control strain were averaged. The results are shown as bars, and standard deviations are indicated as lines. (C) Summary of the mutational analysis indicating the region (EXP) essential for FOB1-dependent rDNA repeat expansion. The locations of segments A to G as well as probes 1, 2, and 3 used for hybridization are shown together with pertinent restriction sites in this region.

FIG. 6

Effects of NTS1 mutations on RFB activity analyzed by 2D gel electrophoresis. DNA was prepared from FOB1 transformants of NTS1 substitution mutants (A to G) and FOB1 and vector transformants (panels FOB1 and fob1, respectively) derived from the control strain, TAK201. DNA was then digested with BglII and subjected to 2D agarose gel electrophoresis followed by Southern hybridization using a rDNA probe (probe 3 in Fig. 5C). Spots indicated by arrowheads show the accumulation of Y-shaped molecules at RFB sites. A schematic diagram of the positions of various Y-shaped replication intermediates is shown as a Y-arc in the bottom right panel.