Ribosomal DNA Replication Fork Barrier and HOT1 Recombination Hot Spot: Shared Sequences but Independent Activities

Abstract

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3 of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).The ribosomal DNA (rDNA) locus in the yeast Saccharomyces cerevisiae consists of 9.1-kb tandem repeats with the 35S rRNA gene, the much smaller 5S rRNA gene, and two nontranscribed spacer (NTS) regions (see Fig. 1) (see references 29 and 22 for reviews of sequence elements in the NTS). NTS2, located between the 5Ј ends of the two genes, contains the promoter for the 35S rRNA gene, a weak origin of replication named the rDNA ARS, and sequences essential for the cisacting mitotic recombination hot spot HOT1. The 35S RNA polymerase I transcriptional enhancer lies in NTS1 near the 3Ј end of the 35S gene. NTS1 also contains sequences important for the polar arrest of replication forks (replication fork barrier [RFB]) and HOT1. The extent of sequence overlap and the interdependence of these two events in DNA metabolism are unknown.The rDNA RFB was first identified in S. cerevisiae, when high-resolution two-dimensional (2D) gel electrophoresis revealed two closely spaced sites where forks arrest (2), herein called RFB1 and RFB2. RFBs appear to be a highly conserved feature of rDNAs, with barriers being found at the 3Ј end of the rRNA genes in a number of other organisms (9,21,23,32,36,38). The yeast RFBs efficiently block replication forks traveling in the direction opposite to 35S transcription, together impeding ϳ90% of encountered forks (2). Fork arrest is not a consequence of transcription per se, since replication forks still arrest at the RFB in cells lacking functional RNA polymerase I (2). The RFB sequences are also not inherently difficult to replicate (2), and thus fork arrest is thought to result from the binding of proteins at the RFB sequences. A protein-mediated mechanism of fork arrest in the rDNA RFB has also been implicated in peas and Tetrahymena thermophila (24, 37) and reported to involve the transcription-terminating factor TTF-I in mice and humans (8,23).HOT1 sequences from the rDNA, when assayed at ectopic sites in the genome, stimulate mitotic homologous recombination between intra-and interchromosomal repeats (...

Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing

“…Interestingly, recombination rates are significantly lower than would be expected for such a large and repetitive locus, suggesting that recombination is somehow suppressed (Petes 1980). Furthermore, recombination levels are reduced despite the presence of several sequence elements within rDNA that can stimulate recombination (Keil and Roeder 1984;Voelkel-Meiman et al 1987;Brewer and Fangman 1988;Kobayashi et al 1992Huang and Keil 1995;Gruber et al 2000;Ward et al 2000). These findings suggest that both positive and negative regulatory mechanisms have evolved to properly control recombination levels of rDNA.…”

Yeast RNA Polymerase I Enhancer Is Dispensable for Transcription of the Chromosomal rRNA Gene and Cell Growth, and Its Apparent Transcription Enhancement from Ectopic Promoters Requires Fob1 Protein

“…As for cis elements required for RFB activities, the work by Ward and coworkers (38) showed that some DNA elements are shared with those required for HOT1 activity, but others are required for one activity but not for the other. Although RFB activities were demonstrated using plasmids carrying cloned DNA containing the E element or the RFB region (3,13,38), the observed activities were reported to be very weak compared to the activities in the chromosomal context (3). This situation resembles that observed for transcription of a reporter gene; transcription of a Pol I reporter on a plasmid was reported to be much weaker relative to transcription of the same reporter integrated into the chromosomal rDNA repeats (16).…”

“…In fact, based on their discovery of the FOB1 gene, which is required for both RFB activity and HOT1 activity, this model was proposed by Kobayashi and Horiuchi (14) as an alternative to the original explanation of HOT1 activity being solely a consequence of high-level transcription from the Pol I promoter. However, it was recently demonstrated that HOT1 activity can take place in the absence of replication fork blocking (38). Thus, the reason why both HOT1 and RFB activities require FOB1 has become a challenging question.…”

A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis

“…In both bacteria, the numerous terminator (Ter) 1 sites are clustered in the terminal region of the chromosome and are arranged so as to control the location of replication fork fusion at the end of a round of replication (for a review see Ref. 10).…”

“…They have roles in a variety of tasks, including the coordination of DNA replication and transcription at eukaryotic rDNA loci (1,2), regulation of mating-type switching in yeast by strand-specific imprinting during replication (3,4), and participation in the final stages of bacterial chromosome replication and partitioning (5).…”