TYPE-I ELEMENTS MEDIATE REPLICATION FORK PAUSING AT CONSERVED UPSTREAM SITES IN THE TETRAHYMENA-THERMOPHILA RIBOSOMAL DNA MINICHROMOSOME

Two-dimensional gel electrophoresis was used to study replication of t he 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 th ree prominent replication fork pausing sites (RFPs) located in or imme diately adjacent to nucleosome-free regions of the 5' NTS. Pausing at these sites is dramatically diminished during replication in Escherich ia coli, suggesting that chromatin organization or Tetrahymena-specifi c proteins may be required. A conserved tripartite sequence was identi fied at each pausing site. Mutations in type I elements diminish pausi ng at proximal RFPs. Hence, type I elements, previously shown to contr ol replication initiation, also regulate elongation of existing replic ation forks. Studies with rDNA transformants revealed a strong directi onal bias for fork pausing. Strong pausing only occurred in forks movi ng toward the rRNA-coding region, We propose that fork pausing in the 5' NTS evolved to synchronize replication and transcription of the dow nstream rRNA genes.