DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

Typically, only a fraction of the >= 600 ribosomal RNA (rRNA) gene copies in human cells are transcriptionally active. Expressed rRNA genes coalesce in specialized nuclear compartments - the nucleoli - and are believed to replicate during the first half of S phase. Paradoxically, attempts to visualize replicating rDNA during early S phase have failed. Here, I show that, in human (HeLa) cells, early-replicating rDNA is detectable at the nucleolar periphery and, more rarely, even outside nucleoli. Early-replicated rDNA relocates to the nucleolar interior and reassociates with the transcription factor UBF, implying that it predominantly represents expressed rDNA units. Contrary to the established model for active gene loci, replication initiates randomly throughout the early-replicating rDNA. By contrast, mostly silent rDNA copies replicate inside the nucleoli during mid and late S phase. At this stage, replication origins are fired preferentially within the non-transcribed intergenic spacers (NTSs), and ongoing rDNA transcription is required to maintain this specific initiation pattern. I propose that the unexpected spatial dynamics of the early-replicating rDNA repeats serve to ensure streamlined efficient replication of the most heavily transcribed genomic loci while simultaneously reducing the risk of chromosome breaks and rDNA hyper-recombination.