The Human RNA Polymerase I Transcription Terminator Complex Acts as a Replication Fork Barrier That Coordinates the Progress of Replication with rRNA Transcription Activity

Abstract

In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes.T he rRNA gene, ribosomal DNA (rDNA), encodes RNA components of ribosomes. In the human genome, there are ϳ400 copies of rDNA encoding the 47S pre-rRNA. These copies are distributed over five clusters of tandemly repeated rDNA on the short arms of acrocentric chromosomes 13, 14, 15, 21, and 22. To meet the vast demand for cellular ribosomes in proliferating human cells, rDNA is heavily transcribed by RNA polymerase I (Pol I). The transcription activity of Pol I fluctuates during the cell cycle; in S phase, the activity is especially vigorous (1). Since both replication and transcription can occur in the same region on the genomic DNA, cells require mechanisms that coordinate these processes.The replication fork barrier (RFB) site near the 3= end of the pre-rRNA coding region has been identified in many organisms, including yeasts, plants, frogs, and mammals (2). In these organisms, with the exception of humans, the RFB predominantly inhibits progression of the replication fork in the opposite direction to pre-rRNA transcription (head-on direction), whereas replication in the same direction (codirection) is not obstructed. Therefore, it is assumed that the RFB arrests the replication fork before it enters the coding region from downstream and thereby prevents the replication fork from colliding with pre-rRNA transcription. In contrast, RFBs in humans are reported to be bidirectional (3).The RFB is formed by a tight complex between certain DNA sequences and proteins that bind to these elements. Fob1 in budding yeast (Saccharomyces cerevisiae) is the best characterized RFB binding protein (4, 5). Deletion of FOB1 allows the replication fork to enter the 35S pre-rRNA coding region from the downstream direction (6, 7). However, when normal numbers of rDNA copies are present, collis...

Rescue from replication stress during mitosis

“…Studies on budding yeast rDNA have identified the existence of natural Replication Fork Barriers (RFBs), replication pause sites defined by specific DNA sequences that bind to non-nucleosomal proteins, blocking replication fork progression in the opposite direction to transcription and acting as recombinational hotspots 65-67 . RFB-mediated mechanisms at rDNA loci to block replication in case of converging transcription, preventing head-on collisions between replication and transcription, have been identified in many organisms, including murine and human cells 64,68,69 . Centromeres are also loci that are difficult to replicate due to their heterochromatic nature and richness in repetitive sequences 70 .…”

Regulation of DNA Replication through Natural Impediments in the Eukaryotic Genome

“…Sal boxes recruit TTF-1, the mammalian ortholog of S. pombe Reb1, which is involved in termination of pre-mRNA transcription [64]. As is the case for S. pombe Swi1 and S. cerevisiae Tof1, human Timeless is required for replication fork arrest at RFBs to coordinate the progression of replication with transcription activity in HeLa cells (Figure 2C) [65]. Thus, the role of the FPC at these pausing sites is conserved between yeast and mammalian cells.…”

“…Deletion of both tof1 and rrm3 , restores pausing to a level significantly higher than that of the wild-type cells [68]. Furthermore, Timeless–Tipin homologs in S. cerevisiae, S. pombe , and humans control the RFBs of rDNA genes in order to prevent collisions between transcription and replication complexes during ribosomal gene transcription [39,65,68,224], suggesting the general role of Timeless–Tipin in preventing genome instability at the interface of DNA replication and transcription (Figure 7). …”

Nucleolar organizer regions: genomic ‘dark matter’ requiring illumination

“…An RFB has also recently been described in the human rDNA repeat (Akamatsu and Kobayashi 2015), and IGS transcripts have been observed in humans and mice in response to various nucleolar stresses or replication senescence (Audas et al 2012;Bierhoff et al 2014). The degree to which the model proposed for yeast can be extended to human cells is an open question.…”

“…The IGS houses gene promoters and regulatory elements, such as spacer promoters and repetitive enhancer elements, which control pre-rRNA synthesis (Goodfellow and Zomerdijk 2013). The IGS also contains replication origins and replication fork barriers (RFBs) that prevent collisions between the replication and transcription machineries (Brewer et al 1992;Kobayashi et al 1992;Akamatsu and Kobayashi 2015). Finally, under certain circumstances, such as stress, loss of repressive chromatin modifications, replicative senescence, or aging, the IGS can be transcribed by RNA Pol II (Earley et al 2010;Audas et al 2012;Saka et al 2013;Bierhoff et al 2014).…”