DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.

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Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.

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Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.