Identification of physiological function of the replication fork blocking protain, Fob1, in yeast Saccharonmyces cerevisiae.

点击翻译按钮获取中文摘要

In most eukaryotic organisms, the ribosomal RNA genes (rDNA) are clustered in long tandem repeats on one or a few chromosomes. The total number of these chromosomal rDNA repeats appears to be maintained at a level appropriate for each organism, thereby indicating that there are some mechanisms to maintain the copy number.The replication fork barrier site (RFB) is 〜100 by DNA sequence located near the 3'-end of rDNA in the yeast, S. cerevisiae. The RFB inhibits the replication fork in the direction opposite to rDNA transcription. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the rDNA, including amplification of rDNA. Therefore, Fob1-dependent RFB activity is thought to result in a recombination hot-spot. However, there has been no direct evidence that Fob1p binds to the RFB sequence. In this study, we found Fob1p directly binds to the RFB, and the DNA seems to wrap around the protein. A predicted zinc finger motif identified in Fob1p was shown to be essential for the RFB binding, replication fork blocking and rDNA recombination activities. These findings implicate Fob1p as the central player in replication fork blocking by binding directly to the DNA and then mediating the blocking of the replication fork.Additionally, it is known that mutations in SIR2 increase instability of rDNA repeats. Sir2p is a NAD-dependent histone deacetylase and is required for gene silencing of Pol II transcription in the rDNA, silent mating type loci and telomeres. We found a significant decrease in the association of the cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Δ relative to SIR2 strains. We concluded that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.