Study on amplification mechanism of ribosomal RNA gene in eucaryotes.

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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.