Topological stress triggers difficult-to-repair DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment

PML, a multifunctional protein, plays a crucial role in forming PML nuclear bodies, which are involved in various cellular processes, including stress responses. Under specific conditions, PML associates with nucleoli, forming PML nucleolar associations (PNAs). However, the stimuli leading to PNAs formation are unknown. Here we investigated these stimuli by exposure of cells to various genotoxic stresses. We reveal that the most potent inducers of PNAs share the ability to inhibit topoisomerases and RNA polymerase I. The inhibition of DNA double-strand break (DSB) repair augmented the occurrence of PNAs linking the stimulus for PNAs formation to unresolved DNA damage. The most potent treatment, doxorubicin, introduced DSBs into the rDNA locus. PNAs co-localized with damaged rDNA, sequestering it from active nucleoli. Using rDNA locus cleavage by I-PpoI, we proved that rDNA damage is a potent PNAs-inducing stimulus. Blocking homology-directed DSB repair (HDR), but not non-homologous end-joining (NHEJ) enhanced PNAs formation, identifying HDR as a PNAs modulator. Our findings have implications for genome stability and diverse diseases and indicate that PNAs form when difficult-to-repair rDNA DSBs occur in nucleoli, highlighting the interplay between the PML PNAs and rDNA alteration caused by deficiencies in topoisomerases, inhibition of RNAPI, and rDNA DSBs destined for HDR.Competing Interest StatementThe authors have declared no competing interest.53BP1p53,binding protein 15-FU5-fluorouracilALTalternative lengthening of telomeresAMDactinomycin DAPBALT-associated PML-NBsAPLacute promyelocytic leukemiaBrdUbromodeoxyuridineCPTcamptothecinCK2casein kinase 2DNA PKDNA-dependent protein kinaseDSBdouble-strand breakhCMVhuman cytomegalovirusHDRhomologous recombination-directed repairhMSChuman mesenchymal stem cellsHRhomologous recombinationHUhydroxyureaIFNγinterferon gammaimmuno-FISHimmunofluorescence-in situ hybridizationIRionizing radiationNCnon-targeting controlNHEJnon-homologous end joiningNORnucleolar organizing regionPMLpromyelocytic leukemia proteinPML-NBspromyelocytic leukemia nuclear bodiesPML-NDSPML nucleolus-derived structurePNAsPML-nucleolar associationspre-rRNAprecursor ribosomal RNARARαretinoic acid receptor alpharDNAribosomal DNARNAiRNA interferenceRNAPIRNA polymerase IRPE-1hTERTtelomerase-immortalized human retinal pigment epithelial cellsSIMSUMO-interacting motifsiRNAsmall interfering RNATDP2tyrosyl-DNA phosphodiesterase 2TOP1topoisomerase 1TOP2topoisomerase 2TPTtopotecanUBFupstream binding factorγH2AXhistone H2AX phosphorylated on serine 139.