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的副本编号得到了很好的控制。例如，即使数字由于某种原因大大减少，它也会自主恢复到原始级别。然而，该机制仍未解决。为了阐明RFB（复制叉屏障）位置的DNA复制叉阻滞事件的生理功能，该场所位于每个rDNA单元中，我们隔离了fork块（命名为fob1）突变体，并意外地发现了RDNA的出乎意料。我们将这种奇怪的表型解释为意味着在RFB站点被逮捕的复制叉上，在任何一个姐妹染色剂上发生了双链破裂（DS破裂）。所得的DS端与相反的姊妹染色剂（不等重组）上的背面或向前rDNA单位不平等地重组，导致rDNA的拷贝数增加或减少。从这种情况下，我们开始了该研究项目，并获得了以下结果。（1）FOB1蛋白直接结合RFB位点，所得的FOB1-RFB复合物阻止了复制叉的进展，（2）FOB1基因作为rDNA区域特异性重组者的表现，（3）除了trans因素，fob1蛋白，命名为exp exp expenion（cis-exp）的位置（在cis expection expection expection expection necy necy expection）necion（suestion expe）（约为400 bp），这是rfb的所需区域。放大，（4）最后，SIR2是一个沉默基因，被称为重组的抑制剂以及rDNA区域的polii依赖性转录。因此，在SIR2突变体中，RDNA之间的重组得到了增强。我们发现，在SIR2条件下，不平等但不相等的重组会增强，并且这种特异性增强不平等的重组来自凝聚力的丧失，这将两个姐妹 - 染色质通过rDNA重复的间隔区联系在一起。