Study on amplification mechanism of ribosomal RNA gene in eucaryotes.

真核生物核糖体RNA基因扩增机制研究。

基本信息

批准号：
13480234
负责人：
HORIUCHI Takashi
金额：
$ 9.6万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Eukaryotic ribosomal RNA genes (the rDNA) are a typical repeated gene. The copy number of rDNA is well controlled. For example, even if the number drastically decreases by some reason, it recovers autonomously to the original level. The mechanism, however, had remained unsolved.In order to elucidate the physiological function of the DNA replication fork blocking event at RFB (replication fork barrier) site, which is located in each rDNA unit, we isolated fork block-less (named fob1) mutants and found unexpectedly that amplification or contraction of rDNA did not occur in the mutants. We interpreted this strange phenotype to mean that a double strand break (ds-break) occurs, on either sister-chromatid, at a replication fork arrested at the RFB site. The resulting ds-end recombines unequally with a back-or a forward-rDNA unit on the opposite sister-chromatid (unequal recombination), leading to an increase or decrease in the copy number of the rDNA, respectively. From this situation, we started this research project, and the following results were obtained. (1) Fob1 protein binds the RFB site directly and the resulting Fob1-RFB complex blocks the progress of the replication fork, (2) the FOB1 gene behaves as an rDNA region specific recombinator, (3) in addition to the trans-factor, Fob1 protein, a cis-element, named the EXP region (about 400 bp) within which the RFB site is located, was identified to be required for rDNA amplification, (4) finally, SIR2 is a silencing gene which is known to act as a suppressor of recombination as well as of PolII dependent transcription in the rDNA region. Thus, in a sir2 mutant, recombination between rDNAs is enhanced. We found that under the sir2 condition, unequal, but not equal recombination is enhanced and this specific enhancement of unequal recombination comes from loss of cohesion, which links the two sister-chromatids together via the spacer region of the rDNA repeats.

真核核糖体RNA基因（rDNA）是典型的重复基因。 rDNA的拷贝数得到很好的控制。例如，即使由于某种原因数量大幅减少，也会自动恢复到原来的水平。然而，其机制仍未解决。为了阐明位于每个rDNA单元中的RFB（复制叉屏障）位点的DNA复制叉阻断事件的生理功能，我们分离了无叉块（命名为fob1）意外地发现，突变体中并没有发生rDNA的扩增或收缩。我们将这种奇怪的表型解释为意味着在任一姐妹染色单体上，在 RFB 位点处捕获的复制叉处发生双链断裂（ds-断裂）。由此产生的 ds 末端与相对姐妹染色单体上的反向或正向 rDNA 单位不相等地重组（不相等重组），分别导致 rDNA 拷贝数的增加或减少。基于这种情况，我们启动了本研究项目，并取得了以下成果。 (1) Fob1 蛋白直接结合 RFB 位点，产生的 Fob1-RFB 复合物阻断复制叉的进程，(2) FOB1 基因充当 rDNA 区域特异性重组子，(3) 除了反式因子之外， Fob1 蛋白是一种顺式元件，被命名为 EXP 区域（约 400 bp），RFB 位点位于该区域内，被鉴定为 rDNA 扩增所必需的，（4）最后，SIR2 为一种沉默基因，已知可作为重组以及 rDNA 区域中 PolII 依赖性转录的抑制因子。因此，在 Sir2 突变体中，rDNA 之间的重组得到增强。我们发现，在sir2条件下，不等但不相等的重组得到增强，这种不等重组的特定增强来自内聚力的丧失，内聚力通过rDNA重复序列的间隔区将两个姐妹染色单体连接在一起。