Study on amplification mechanism of ribosomal RNA gene in eucaryotes.

真核生物核糖体RNA基因（rDNA）是一种典型的重复基因。rDNA的拷贝数得到了很好的控制。例如，即使数量由于某种原因急剧减少，它也会自动恢复到原始水平。为了阐明位于每个rDNA单元中的RFB（replication fork barrier）位点的DNA复制叉阻断事件的生理功能，我们分离了无复制叉阻断（fork block-less，简称fob 1）突变体，意外地发现突变体中没有rDNA的扩增或收缩。我们解释这种奇怪的表型意味着双链断裂（ds-break）发生在任一姐妹染色单体上，在RFB位点的复制叉处。由此产生的ds-末端与相对姐妹染色单体上的后向或前向rDNA单元不相等地重组（不相等重组），分别导致rDNA拷贝数的增加或减少。从这种情况出发，我们开始了这项研究项目，并取得了以下成果。(1)Fob 1蛋白直接结合RFB位点，产生的Fob 1-RFB复合物阻断复制叉的进程，（2）FOB 1基因表现为rDNA区域特异性重组子，（3）除了反式因子，Fob 1蛋白，顺式元件，称为EXP区域（约400 bp），其中RFB位点位于其中，被鉴定为rDNA扩增所需，（4）最后，SIR 2是一种沉默基因，已知其在rDNA区域中充当重组以及PolII依赖性转录的抑制子。因此，在sir 2突变体中，rDNAs之间的重组增强。我们发现，sir 2条件下，不平等的，但不平等的重组增强，这种特殊的增强不平等的重组来自失去凝聚力，这两个姐妹染色单体通过rDNA重复的间隔区连接在一起。

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.