A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage

Abstract

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 fromnucleoli. 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 thatHR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.