Type I Elements Mediate Replication Fork Pausing at Conserved Upstream Sites in the Tetrahymena thermophila Ribosomal DNA Minichromosome

Abstract

Two-dimensional gel electrophoresis was used to study replication of the Tetrahymena thermophila ribosomal DNA (rDNA) minichromosome. During vegetative growth, the rDNA is replicated exclusively from origins in the 5 nontranscribed spacer (NTS). Whereas replication fork movement through the rest of the chromosome appears to be continuous, movement through the 5 NTS is not. Replication forks arrest transiently at three prominent replication fork pausing sites (RFPs) located in or immediately adjacent to nucleosome-free regions of the 5 NTS. Pausing at these sites is dramatically diminished during replication in Escherichia coli, suggesting that chromatin organization or Tetrahymena-specific proteins may be required. A conserved tripartite sequence was identified at each pausing site. Mutations in type I elements diminish pausing at proximal RFPs. Hence, type I elements, previously shown to control replication initiation, also regulate elongation of existing replication forks. Studies with rDNA transformants revealed a strong directional bias for fork pausing. Strong pausing only occurred in forks moving toward the rRNA-coding region. We propose that fork pausing in the 5 NTS evolved to synchronize replication and transcription of the downstream rRNA genes.

A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome

“…4A (Fork Pause). The role and cause of these pauses have not yet been determined (31). Figure 4B shows the 2D gel patterns obtained from homozygous C3 and homozygous C3-rmm3 cells.…”

“…4B and data not shown) and in log-phase cells (data not shown). The repeat 2 pause site has been mapped to nt 1075 (Ϯ35 nt) (31). This site is roughly 750 nt upstream of the rmm3 base change.…”

“…In vegetatively growing cells, the physical origin of replication is within the 1.9-kb 5Ј nontranscribed spacer (NTS) of the rRNA gene and localizes to roughly the repeat 1-repeat 2 region (Fig. 1B) (6,31). Repeats 1 and 2 consist of two Ͼ90% homologous, ϳ400-bp tandem direct repeats.…”

“…Wild type C3 491-1a and C3-rmm3 cells were grown in 200 ml of 2% PPYS to a density of 2 ϫ 10 5 to 3 ϫ 10 5 cells/ml for log-phase cell samples. In addition, cells were starved for 20 h and refed to synchronize them and to enrich for rDNA replicating intermediates (6,11,31). Aliquots (100 ml) were processed for DNA at 70, 80, and 90 min after refeeding.…”

“…Aliquots (100 ml) were processed for DNA at 70, 80, and 90 min after refeeding. DNA was prepared as described previously (31) except that proteinase K was added to a final concentration of 8 mg/ml. Forty micrograms of DNA in a 1-ml volume was digested with a 10-fold excess of HindIII (20 U/liter) for 3 h at 37°C.…”