Abstract
The genes encoding ribosomal RNA are the most abundant in the eukaryotic genome. They reside in tandem repetitive clusters, in some cases totaling hundreds of copies. Due to their repetitive structure, ribosomal RNA genes (rDNA) are easily lost by recombination events within the repeated cluster. We previously identified a unique gene amplification system driven by unequal sister-chromatid recombination during DNA replication. The system compensates for such copy number losses, thus maintaining proper copy number. Here, through a genome-wide screen for genes regulating rDNA copy number, we found that the rtt109 mutant exhibited a hyper-amplification phenotype (∼3 times greater than the wild-type level). RTT109 encodes an acetyl transferase that acetylates lysine 56 of histone H3 and which functions in replication-coupled nucleosome assembly. Relative to unequal sister-chromatid recombination-based amplification (∼1 copy/cell division), the rate of the hyper-amplification in the rtt109 mutant was extremely high (>100 copies/cell division). Cohesin dissociation that promotes unequal sister-chromatid recombination was not observed in this mutant. During hyper-amplification, production level of extra-chromosomal rDNA circles (ERC) by intra-chromosomal recombination in the rDNA was reduced. Interestingly, during amplification, a plasmid containing an rDNA unit integrated into the rDNA as a tandem array. These results support the idea that tandem DNA arrays are produced and incorporated through rolling-circle-type replication. We propose that, in the rtt109 mutant, rDNA hyper-amplification is caused by uncontrolled rolling-circle-type replication.
Highlights
The ribosome is an abundant macromolecular protein-RNA complex that translates mRNA into protein
Identification of mutants with high rDNA copy number To elucidate the mechanism which regulates rDNA copy number, we measured the size of rDNA in the,4,800 mutants that comprise the yeast deletion library (S288c genetic background) by pulsed-field gel electrophoresis (CHEF, Figure S1)
We identified eight mutants with higher-than-usual copy numbers (,150), and presumed that they were defective in copy number regulation (Table 1)
Summary
The ribosome is an abundant macromolecular protein-RNA complex that translates mRNA into protein. In Saccharomyces cerevisiae, ribosomal proteins (RP) account for approximately 50% of total protein and ribosomal RNA (RNA) represents approximately 80% of total RNA [1]. While the amount of RP increases during translation from mRNA, the amount of rRNA is dependent on transcription level. To meet this huge biosynthetic demand for rRNA in eukaryotic cells, the rRNA genes are present in hundreds of copies and are transcribed by a highly efficient RNA polymerase, RNA pol I [1]. The highly repetitive structure of the rDNA makes it fragile and vulnerable to loss of copies following homologous recombination events within the repeat
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