Repeated sequences and genes are extensively distributed throughout the genomes of many organisms. A common feature of these repeats is instability; the copy number of repeats frequently fluctuates, and they are exposed to the risk of contraction. However, the mechanisms responsible for the fluctuation of repeat copy number and for the maintenance of the repeats remain largely unknown. The eukaryotic rRNA gene (rDNA) family is a typical repeated structure. The genes are generally clustered in long tandem repeats on one or several loci in most eukaryote genomes. In the yeast Saccharomyces cerevisiae
, 200 copies on average are tandemly arrayed in a central position on the longest chromosome, chromosome XII. A single rDNA unit (9.1 kb) comprises a 35S rRNA coding region which is transcribed by RNA polymerase I, the 5S rRNA coding region transcribed by RNA polymerase III, and two nontranscribed spacers (NTS), i.e., NTS1 and NTS2 (see Fig. ). The replication origin (ARS) and the replication fork barrier (RFB) are located within NTS2 and NTS1, respectively (5
). The whole length of the tandem array extends ~1.9 Mb.
FIG. 5. FOB1-dependent condensin association with the RFB site in the rDNA. (A) The structure of the rDNA tandem array is shown above, and an enlarged single (9.1-kb) copy is shown below. The positions of the PCR fragments used for the ChIP assay are indicated (more ...)
One system that maintains the average copy number of the rDNA long tandem array has been reported in recent years. High frequencies of recombination events and expansion/contraction of the repeats are observed for FOB1+
strains, whereas deletion of FOB1
) eliminates both events (14
). Furthermore, DNA strand break formation around the RFB site was observed to be FOB1
is therefore thought to be required for the maintenance of average copy number by increasing or decreasing copy number through the regulation of recombination events between the repeats. In Δfob1
cells, surprisingly, the rDNA tandem array is still maintained stably without any expansion/contraction. The static rDNA tandem array maintained in Δfob1
suggests that a second, unknown system, one that is different from the FOB1
-stimulated recombinational system, acts to maintain the long tandem repeats. Originally, FOB1
was identified as the gene required for replication fork blocking in the direction opposite to 35S rRNA transcription at the RFB site (26
). Recently, both in vivo and in vitro RFB site binding activity of Fob1p was demonstrated (28
). It is believed that the Fob1p/RFB system is required to avoid a head-to-head collision between replication and transcription of RNA polymerase I (5
) and to contribute in the maintenance of chromosome integrity.
Condensin in S. cerevisiae
is a protein complex consisting of five subunits, Smc2p, Smc4p, Brn1p, Ycs4p, and Ycs5p (Ycg1p), and plays an important role in condensation and segregation of chromosomes during mitosis (23
). All subunits are conserved across the eukaryotes. The two core subunits, Smc2p and Smc4p, belong to the “structural maintenance of chromosomes” family, whose members are involved in various chromosome dynamics; SMC1
encode subunits of cohesin that are required for connection of sister chromatids (35
), and SMC5
are found to work for the segregation of repetitive chromosome regions (48
). Condensin is essential for cell growth and has been reported to play a role in mitosis as well as in interphase (2
). Cytological observations in S. cerevisiae
demonstrated that condensin binds throughout the genome during interphase and is concentrated to the nucleolus in anaphase (4
). Recently, a novel finding was reported. Although the majority of sister chromatid disjunctions are triggered by cleavage of cohesin during metaphase/anaphase transition (49
), sister chromatids on the long arm of chromosome XII that contain the rDNA repeats are still held together despite cohesin destruction. The disjunction of sister chromatids of this locus occurs in mid-anaphase, long after cohesin destruction, and condensin is required for this step (13
). However, the mechanisms by which the sister chromatids at the rDNA locus are held together and the role of condensin in chromosome segregation and condensation remain largely unknown.
In this study, we attempted to understand the putative second, unknown system by which the long rDNA arrays are maintained by isolating mutants carrying an unstable, shortened rDNA tandem array in a Δfob1 background. We found that condensin mutants showed severe contraction of rDNA tandem array and slow growth in Δfob1 cells but not in FOB1+ cells. Chromatin immunoprecipitation (ChIP) showed that condensin complex was loaded onto the RFB site in a FOB1-dependent fashion during S phase and remained associated until anaphase. These results indicate that there is a novel system to prevent contraction of long rDNA tandem array securely through FOB1-dependent condensin complex association to the rDNA.