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

在大多数真核生物中，核糖体RNA基因（rDNA）在一条或几条染色体上以长串联重复序列聚集。这些染色体rDNA重复序列的总数似乎维持在适合于每种生物的水平上，从而表明存在一些维持拷贝数的机制。啤酒。RFB在与rDNA转录相反的方向上抑制复制叉。基因FOB1是这种RFB活性所必需的。FOB1也是rDNA重组所必需的，包括rDNA的扩增。因此，Fob 1依赖性RFB活性被认为导致重组热点。然而，没有直接证据表明Fob1p与RFB序列结合。在这项研究中，我们发现Fob1p直接与RFB结合，DNA似乎包裹着蛋白质。Fob1p中的锌指基序对RFB结合、复制叉阻断和rDNA重组活性至关重要。这些发现暗示Fob1p是复制叉阻断的核心参与者，它直接与DNA结合，然后介导复制叉的阻断。此外，已知SIR2的突变增加了rDNA重复序列的不稳定性。Sir2p是NAD依赖性组蛋白脱乙酰酶，并且是rDNA中Pol II转录、沉默交配型基因座和端粒中的基因沉默所需的。我们发现，相对于SIR2菌株，sir2Δ中的粘附素亚基Mcd1p（Scc1p）与rDNA的结合显著减少。我们的结论是，SIR2防止不平等的姐妹染色单体重组，可能是通过形成特殊的粘蛋白结构，没有显着的影响，在个别rRNA基因的重组事件。

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.