Binding of the Replication Terminator Protein Fob1p to the Ter Sites of Yeast Causes Polar Fork Arrest

Abstract

Fob1p protein has been implicated in the termination of replication forks at the two tandem termini present in the non-transcribed spacer region located between the sequences encoding the 35 S and the 5 S RNAs of Saccharomyces cerevisiae. However, the biochemistry and mode of action of this protein were previously unknown. We have purified the Fob1p protein to near-homogeneity, and we developed a novel technique to show that it binds specifically to the Ter1 and Ter2 sequences. Interestingly, the two sequences share no detectable homology. We present two DNA replication in many prokaryotes is often terminated at sequence-specific replication termini (Ter sites). Replication terminator proteins specifically bind to the Ter sites and arrest forks in an orientation-specific mode with respect to the replication origins (1). In many eukaryotes, although every replicon in the multiorigin chromosomes does not have specific Ter sites, such sites are present in the nontranscribed spacer sequences in the rDNA from yeast to man (1, 2).In Escherichia coli and Bacillus subtilis, the Ter sites bind to replication termination proteins, called Tus and RTP, respectively, that are contrahelicases (3, 4) and impede replication fork movement not only by binding to the Ter sites but also by protein-protein interaction between the helicase and the terminator protein (5). The rDNAs of Saccharomyces cerevisiae (6, 7), Schizosaccharomyces pombe (8), Xenopus (9), mouse (10), pea (11), and humans (12) have similar replication fork arrest systems located in their nontranscribed spacer elements. In S. pombe, replication fork arrest has been shown to occur at and near the mating type switch locus (13-15).The rDNA locus of S. cerevisiae (Fig. 1) consists of 100 -200 tandem copies of 9.1-kb rDNA units present in chromosome number XII. Each unit of the rDNA consists of a 35 S rRNA and a 5 S rRNA gene that transcribe in opposite directions. A nontranscribed spacer called NTS2 containing an origin of replication called ARS 1 (autonomously replicating sequence) is located between the genes encoding the 35 S and the 5 S RNAs (6, 16).The two replication forks initiated at the ARS face unequal fate. The rightward moving fork moves through the 35 S rDNA in the same direction as that of transcription until it meets the fork coming from the opposite direction. But the leftward moving fork, after passing through the 5 S rRNA gene, is arrested at two Ter sites located in the nontranscribed spacer called NTS1 and is thus prevented from entering the 35 S rRNA gene. The two Ter sites (known as replication fork barrier or RFB sites) located in NTS1 arrest replication forks in an orientationdependent manner (6, 7). The Ter1 and Ter2 sites are separated from each other by a few nucleotides and can be further separated by inserting foreign DNA between them without affecting their activities (7).The Ter sites in prokaryotes and eukaryotes are recombinogenic (2). The yeast rDNA also contains a recombination hot spot HOT1 that consists of two elements called enhanc...

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…The left edge of the tandem array terminates precisely within the RFB of the terminal NTS1 element (Figure 11) (Bairwa et al 2010). Fob1 typically binds two cis-acting sequences within the RFB, known as Ter1 and Ter2 (Kobayashi 2003;Mohanty and Bastia 2004). Only Ter2 remains at the left flank, resulting in attenuated recruitment of RENT and suboptimal silencing (Bairwa et al 2010;Buck et al 2016).…”

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

“…Considerable progress in the characterization of replication termination systems has been made with those from Bacillus subtilis and Escherichia coli (6,7), and this has recently aided the study of analogous eukaryotic systems (4,8,9). 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.…”

Site-Directed Mutagenesis and Yeast Reverse 2-Hybrid-Guided Selections to Investigate the Mechanism of Replication Termination

“…Net1 recruits the histone deacetylase Sir2 onto Fob1 by direct protein-protein interaction between Net1 and Sir2 on one hand and between Net1 and Fob1 on the other, and loads Sir2 near the Ter sites. This process, as noted above, causes silencing of rDNA and prevents unwanted recombination (Bairwa et al, 2010;Mohanty and Bastia, 2004). At this time, the detailed mechanism of replication termination in eukaryotes has not been elucidated.…”

“…The second spacer contains a replication ori (ars; see Fig.5). The Ter sites bind to the replication terminator protein called Fob1 (fork blockage) (Kobayashi, 2003;Kobayashi and Horiuchi, 1996;Mohanty and Bastia, 2004). The Fob1 protein bound to Ter sites prevents replication forks moving from right to left from c oll i ding with the strong transc ription of 35S RNA.…”

“…These "pull down" assays are used to confirm the reverse 2-hybrid results. If the interaction of X and Y is necessary for a biological function (e.g., fork arrest at Tus-Ter complex), the X mutants that do not interact with protein Y are then tested by 2-dimensional agarose gel electrophoresis Fangman, 1987, 1988;Mohanty et al, 2006;Mohanty and Bastia, 2004) to determine whether they show the expected biochemical property (in this case, failure to arrest replication forks) (Mulugu et al, 2001). The reverse 2-hybrid approach is a powerful method that can yield mutants that specifically disrupt protein-protein interaction between a pair of known interacting proteins.…”