Inhibition of topoisomerase I prevents chromosome breakage at common fragile sites.
Common fragile sites are loci that preferentially form gaps and breaks on metaphase chromosomes when DNA synthesis is perturbed, particularly after treatment with the DNA polymerase inhibitor, aphidicolin. We and others have identified several cell cycle checkpoint and DNA repair proteins that influence common fragile site stability. However, the initial events underlying fragile site breakage remain poorly understood. We demonstrate here that aphidicolin-induced gaps and breaks at fragile sites are prevented when cells are co-treated with low concentrations of the topoisomerase I inhibitor, camptothecin. This reduction in breakage is accompanied by a reduction in aphidicolin-induced RPA foci, CHK1 and RPA2 phosphorylation, and PCNA monoubiquitination, indicative of reduced levels of single stranded DNA. Furthermore, camptothecin reduces spontaneous fragile site breakage seen in cells lacking ATR, even in the absence of aphidicolin. These data from cultured human cells demonstrate that topoisomerase I activity is required for DNA common fragile site breaks and suggest that polymerase-helicase uncoupling is a key initial event in this process.
Arlt
Martin F
MF
Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA. arltm@umich.edu
Glover
Thomas W
TW
D016590:Aphidicolin / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D019457:Chromosome Breakage / Q000187:drug effects*; D043283:Chromosome Fragile Sites / Q000187:drug effects* / Q000235:genetics*; D004261:DNA Replication / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D053978:Pentacyclic Triterpenes; D010766:Phosphorylation / Q000187:drug effects; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency; D051716:Replication Protein A / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D014315:Triterpenes / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects; D000094062:Betulinic Acid
D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D053978:Pentacyclic Triterpenes; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D059004:Topoisomerase I Inhibitors; D014315:Triterpenes; D016590:Aphidicolin; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; C516794:RPA2 protein, human; D002166:Camptothecin; D000094062:Betulinic Acid