The Replication Fork Barrier Site Forms a Unique Structure with Fob1p and Inhibits the Replication Fork

Abstract

The replication fork barrier site (RFB) is an ϳ100-bp DNA sequence located near the 3 end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.Sites that cause replication fork pausing have been identified in many genomes and are known as replication fork-blocking sites. The replication fork-blocking site was first identified in bacteria, both in plasmids and in the genome. Plasmid R6K and the genome of Escherichia coli each have a replication termination sequence, Ter (for a review, see reference 18). Ter is a conserved 22-bp sequence that inhibits the replication fork in a polar fashion (18). The Bacillus subtilis genome has a counterpart of Ter, called RTS, although there is no similarity between the sequences (5, 34). In E. coli, the tus protein binds specifically to the Ter sequence, and the Tus-Ter complex inhibits the action of DNA helicases to arrest replication (14,15,28,32). Ter is also known as a recombination hot spot that stimulates both DNA double-strand breaks (37) and recombination (16, 17; for a review, see reference 41). Therefore, one possible role of Ter is thought to be maintenance of genome stability.In eukaryotic cells, replication fork-blocking sites have been identified in the rRNA gene repeats (rDNA) from yeast to human cells (for a review, see reference 41). They are called replication fork barriers (RFB) and inhibit replication forks in the direction opposite to rDNA transcription. The RFB has been studied most intensively in the yeast Saccharomyces cerevisiae. S. cerevisiae carries about 150 copies of the rDNA repeats. The RFB is located near the 3Ј end of the 35S rDNA in one of the nontranscribed spacer (NTS) regions (NTS1) (Fig. 1A). In the other NTS region (NTS2), there is an autonomously replicating sequence (ARS) which functions as a replication origin. In the S phase of the cell cycle, replication starts at the ARS bidirectionally and the rightward-moving replication forks are arrested at the RFB (Fig. 1). However, the other forks can go through the RFB site because of RFB polarity, like for Ter in E. coli (4,29). Therefore, one of the biological roles of the RFB is tho...

Sap1p Binds to Ter1 at the Ribosomal DNA of Schizosaccharomyces pombe and Causes Polar Replication Fork Arrest

“…The paradigm that site-specific terminators are required for fork arrest at eukaryotic Ter sites seems to hold true as well, as binding of Fob1p to the rDNA Ter sites is required for polar fork arrest in S. cerevisiae (22,23). Similarly, mammalian TTF-1 mediates fork arrest at the terminators in mouse rDNA (24).…”

The Histone Deubiquitinating Enzyme Ubp10 Is Involved in rDNA Locus Control in Saccharomyces cerevisiae by Affecting Sir2p Association

“…UBP10 loss of function results in a decrease of Sir2p level at both NTS1 and NTS2 that correlates with histone hyperacetylation and, consequently, produces a more open chromatin configuration. Notably, the NTS1 fragment analyzed overlaps the replication fork block (Kobayashi 2003), suggesting that accumulation of ERCs observed in the ubp10D mutant can be ascribed to the reduced extent of Sir2p-dependent silent chromatin required to counteract Fob1p-dependent rDNA recombination at this region.…”

Rates of Recombination in the Ribosomal DNA of Apomictically Propagated Daphnia obtusa Lines

“…There are opposing viewpoints on how rDNA structure will affect recombination rate. For example, the recombination rate in rDNA may be high relative to the genomewide average due to the fact that each primary unit, which is repeated tens to thousands of times, contains a copy of a region containing the replication fork block, which is known to be associated with an increased probability of recombination (Kobayashi 2003). Indeed, selection may favor an elevated rate of recombination in rDNA due to its impact on the homogenization of gene copies along the rDNA array.…”