Conserved features in the mode of replication of eukaryotic ribosomal RNA genes

It was previously shown that a 1.5 kb fragment located in the non-transcribed spacer (NTS) is the earliest replicating region of pea (Pisum sativum) rDNA in synchronized root cells. In the present report the structure of this region was characterized. It contains a cluster of four 11 bp near matches to the Saccharomyces cerevisiae ARS consensus sequence (ACS). These near matches are embedded in an A+T rich domain located upstream from the transcription initiation site. We identified and mapped an intrinsic DNA bending locus 5' to the cluster of near matches. Several eukaryotic origins including the ARS from the budding yeast show very similar structural features. This observation strengthens the notion that pea rDNA replication initiates at or near this region. Replication of the entire pea rDNA repeat was analysed by two-dimensional (2D) agarose gel electrophoresis. The results obtained indicate that only a small fraction of the potential origins is used in each replication round. Forks moving in the direction opposite to rRNA transcription are stalled at a polar replication fork barrier (RFB), which mapped near the 3' end of the transcription unit. Consequently, most of pea rDNA appears to replicate in a unidirectional manner. These results show that the strategy used to replicate pea and yeast rRNA genes is very similar, suggesting that it has been conserved and might be common to most eukaryotes.