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)聚集在一个或几个染色体上的长串联重复序列中。这些染色体rDNA重复序列的总数似乎保持在适合每个生物体的水平，从而表明有一些机制来维持拷贝数。在酵母中，位于rDNA 3‘端附近的DNA序列的复制分叉屏障位置(RFB)为~100。RFB在与rDNA转录相反的方向上抑制复制分叉。FOB1基因是这种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，依赖于Fob1的RFB活性被认为导致了重组热点。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似乎包裹在蛋白质周围。在Fob1p中发现的一个预测的锌指基序对于RFB结合、复制分叉阻断和rDNA重组活动是必不可少的。这些发现暗示Fob1p是复制分叉阻断的中心角色，它直接与DNA结合，然后介导复制分叉的阻断。此外，已知Sir2的突变增加了rDNA重复序列的不稳定性。Sir2p是一种依赖NAD的组蛋白脱乙酰酶，在rDNA、沉默交配型基因座和端粒中，Pol II转录的基因沉默是必需的。我们发现，与Sir2株相比，SIR2Δ中粘附素亚基Mcd1p(Scc1p)与rDNA的关联性显著降低。我们的结论是，Sir2可能通过形成特殊的粘附素结构来防止不平等的姐妹染色单体重组，而对单个rRNA基因内的重组事件没有显著影响。

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.