Nucleolar reorganisation promotes repair of rDNA double strand breaks by homologous recombination throughout the cell cycle.

Abstract

The nucleolus is the largest functional domain within the nucleus and is the site of ribosome biogenesis. It has a distinct structure and houses ribosomal RNA gene (rDNA) transcription, pre-ribosomal RNA (pre-rRNA) processing, and pre-ribosome assembly. The rDNA repeats encode the major rRNA species and are organized into large head-to-tail tandem arrays located at the nucleolar organiser regions (NORs). In humans, the ~300 rDNA repeats are distributed among five NORs on the short arms of the acrocentric chromosomes. In most human cells a majority of NORs, but usually not all, are active, and coalesce to form between 1 and 3 nucleoli. Nucleoli are spatially isolated from the rest of the nucleoplasm by a shell of heterochromatin. The enormous demand for ribosomes by actively growing cells means that rDNA repeats are the most actively transcribed genes in all eukaryotic cells. The resulting vulnerability of rDNA combined with emerging roles for the nucleolus in stress sensing prompted us to investigate the response of nucleoli to the presence of double strand breaks (DSBs) in the rDNA. The 28S rRNA coding sequences contain the 15bp recognition sequence for the homing endonuclease I-PpoI. I have developed a broadly applicable mRNA transfection procedure that can efficiently introduce I-PpoI into a wide variety of human cell lines, thus inducing DSBs specifically within the rDNA repeats. Analysis of how nucleoli deal with DSBs in the rDNA has uncovered a complex response. The presence of DSBs results in activation of ATM and inhibition of transcription. As a direct consequence of transcriptional inhibition, rDNA repeats withdraw from the nucleolar interior to caps located at the nucleolar periphery. Importantly each cap represents the rDNA from a single NOR. Positioning of damaged NORs on the nucleolar surface renders their damaged rDNA accessible to repair factors, normally excluded from the nucleolar interior. Evidence suggests that repair is carried out by homologous recombination (HR), independently of the stage in the cell cycle. This complex nucleolar response highlights the fact that a spectrum of DSB repair mechanisms have evolved to maintain integrity of the genome.