Abstract
Polar replication fork barriers (RFBs) near the 3' end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3' to 5' order. The transcription termination protein reb1 and its two binding sites, present at the 3' end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist.
Highlights
During eukaryotic ribosomal DNA replication, the fork moving opposite to transcription is arrested at replication fork barriers (RFBs) close to the 3Ј end of the coding region [3, 13, 24,25,26,27, 42, 43]
Three independent spots were identified on the descending portion of the simple-Y arc (Fig. 1B), indicating that replication stalled at three alternative sites, called RFB1, RFB2, and RFB3
Fork arrest at two of these RFBs (RFB2 and RFB3) is produced upon binding of the transcription termination protein reb1, in a fashion similar to what has been proposed to occur in mouse [27, 35]
Summary
During eukaryotic ribosomal DNA (rDNA) replication, the fork moving opposite to transcription is arrested at replication fork barriers (RFBs) close to the 3Ј end of the coding region [3, 13, 24,25,26,27, 42, 43]. In mouse rDNA, replication forks stall at the rRNA transcriptional terminator elements known as Sal boxes [27], which are the specific binding sites for transcription termination factor mTTF-1 [12, 21] This protein was able to arrest replication forks in an in vitro replication assay [10, 35]. Contrary to what happens in the mouse, neither the rRNA transcription termination factor Reb1p nor its rDNA binding sequence seems to be involved in the RFB of S. cerevisiae [4, 41] These observations suggest that the molecular mechanism that regulates rDNA replication arrest diverged through evolution. Schizosaccharomyces pombe could be a transitional species in which the mechanisms operating in budding yeast and mammals coexist
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