The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks

Abstract

In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3′ end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1. To identify fork stalling we used high-resolution 2D agarose gel electrophoresis. The results obtained indicated that neighbor DNA sequences and the relative abundance of Fob1 modulate the efficiency of rRFBs to stall replication forks.

Replication fork pausing at protein barriers on chromosomes

“…B). The efficiency of the fork pausing at the RFB–Fob1 binding site was evaluated using high‐resolution 2D agarose gels, which detected transient (not permanent) fork pausing at the RFB, for which the efficiency is dependent on the amount of Fob1 . The molecular mechanism of the fork pausing at the RFB by Fob1 remains unclear.…”

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks

“…Not all the potential Fob1p binding sites actually binds Fob1p, and many forks go through the potential barriers with no trouble. [ 15,46 ] In any case, some forks do stall at the barriers, and the abundance and corresponding intensity of the signal of RIs containing the stalled fork become stronger in 2D gels (Figure 4A). The position of the stronger spot indicates the mass of the RI accumulated.…”

“…The closer the cross to either of the two ends, the electrophoretic mobility increases while it becomes minimal when the cross is at the middle. [ 15 ] It should be noticed that in their original paper, Brewer and Fangman only proposed an interpretation for the various patterns observed. [ 14 ] The verification came later with a combination of 2D gels and electron microscopy.…”

“…The replication forks stalled at only two close conspicuous sites and the intensity of the signal was stronger for the first barrier met by the replicating fork. [ 15 ]…”

“…It was concluded that in the ribosomal DNA of S. cerevisiae , the abundance of Fob1p modulates the efficiency of ribosomal RFBs to stall replication forks. [ 15 ]…”