title: Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition.
abstract: Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.
authors.affiliation: University of North Carolina, Chapel Hill, NC USA. kpalle@med.unc.edu
authors.forename: Komaraiah
authors.initials: K
authors.lastname: Palle
authors.forename: Cyrus
authors.initials: C
authors.lastname: Vaziri
mesh_terms: D002166:Camptothecin / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival; D002843:Chromatin / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052217:Fanconi Anemia Complementation Group A Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D016196:S Phase; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D052217:Fanconi Anemia Complementation Group A Protein; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; D006657:Histones; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; D034741:RNA, Small Interfering; D059004:Topoisomerase I Inhibitors; D044767:Ubiquitin-Protein Ligases; D002166:Camptothecin