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

酿酒酵母中复制叉阻断蛋白 Fob1 的生理功能鉴定。

基本信息

批准号：
14380332
负责人：
KOBAVASHI Takehiko
金额：
$ 8.9万
依托单位：
National Institute for Basic Biology (2004)Okazaki National Research Institutes (2002-2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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.

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聚集成长串联重复。这些染色体 rDNA 重复的总数似乎维持在适合每种生物的水平，从而表明存在。一些维持拷贝数的机制。酿酒酵母中位于 rDNA 3' 端附近的 DNA 序列的复制叉屏障位点 (RFB) 约为 100。 RFB 以与 rDNA 转录相反的方向抑制复制叉。该 RFB 活性需要基因 FOB1，对于 rDNA 中的重组（包括 rDNA 的扩增）而言，FOB1 也是必需的。因此，Fob1 依赖性 RFB 活性被认为是由此产生的。然而，没有直接证据表明 Fob1p 与 RFB 序列结合。在本研究中，我们发现 Fob1p 直接与 RFB 结合。 DNA 似乎包裹着蛋白质。在 Fob1p 中发现的预测锌指基序被证明对于 RFB 结合、复制叉阻断和 rDNA 重组活动至关重要。这些发现表明 Fob1p 通过直接结合而成为复制叉阻断的核心参与者。此外，SIR2 的突变会增加 rDNA 重复序列的不稳定性，Sir2p 是一种 NAD 依赖性组蛋白脱乙酰酶。 rDNA、沉默交配型位点和端粒中 Pol II 转录的基因沉默所需。我们发现，相对于 SIR2 菌株，sir2Δ 中粘连蛋白亚基 Mcd1p (Scc1p) 与 rDNA 的关联显着减少。姐妹染色单体重组，可能是通过形成特殊的粘连蛋白结构，对单个 rRNA 基因内的重组事件没有显着影响。