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. MMS, methyl methanesulfonate; NBS1, nijmegen breakage syndrome protein1; NHEJ, non-homologous end joining; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; Polη, DNA polymerases eta; Polι, DNA polymerases iota; Polκ, DNA polymerases kappa; RFC, replication factor C; RPA, replication protein A; RT, room temperature; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; siRNA, small interference RNA; ssDNA, single-stranded DNA; TBE, tris-boric acid-EDTA (buffer); TLS, translesion DNA synthesis; Top1, DNA topoisomerase I; Top1cc, DNA-topoisomerase I covalent or cleavage complex; Usp1, ubiquitin-specific processing protease 1; UV, ultra violet radiation; WT, wild type ©2 0 1 1 L a n d e s B i o s c i e n c e . D o n o t d i s t r i b u t e .1626 Cell Cycle Volume 10 Issue 10 with TLS, ensuring appropriate processing of bulky DNA lesions. FA is a cancer predisposition syndrome in humans. To date, at least 13 complementation groups of FA have been identified (designated 'FANC' A-M). The FA proteins (FANCs) are thought to work cooperatively in a common pathway to repair interstrand crosslinks. 28 The eight-prote...

Gli1 Protein Regulates the S-phase Checkpoint in Tumor Cells via Bid Protein, and Its Inhibition Sensitizes to DNA Topoisomerase 1 Inhibitors

“…Similar results have also been observed in several independent studies when Chk1 was inhibited in cancer cells (39,72,73). We and others have shown previously that Top1 poisoning by CPT not only induces replication stress but also leads to replication-coupled DSBs because prolonged stabilization of Top1cc by CPT can lead to fork collapse (29,31,37,72,73). Therefore, active replication is important for CPT-induced cytotoxic effects.…”

“…RNA Interference-The siRNAs used in this study were purchased from Dharmacon and transfected using Lipofectamine 2000 (Invitrogen) according to the protocols of the manufacturer and as described previously (37). The siRNAs used in this study were as follows: siControl (37), siGli1-1, siGli1-2, siGli1-5 (49), and siBid (50).…”

“…Although ataxia telangiectasia mutated (ATM)/ Chk2 and ATR/Chk1 kinase-mediated signaling cascades are activated in response to CPT treatment, ATR/Chk1-mediated responses play a predominant role in orchestrating DDR signaling and cell survival (30,(33)(34)(35)(36). These effects can be attributed to the generation of replication stress and replication-coupled DSBs because of Top1 poisoning by CPT (37). The primary response to the replication block is mediated by the activation of ATR-mediated signaling in association with several factors (RPA, ATRIP, claspin, etc.…”

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

“…[122][123][124][125][126] RAD18-RAD6 catalyzes the monoubiquitination of PCNA on K164, a molecular event necessary for DNA polymerase switching during translesion DNA synthesis (TLS).…”

“…122,126 Genetic disruption of RAD18 or RAD18 depletion via siRNA leads to decreased ICL-induced FANCD2/I monoubiquitination and chromatin binding. [122][123][124][125][126] Furthermore, RAD18-RAD6 mediated monoubiquitination of PCNA on K164 is necessary for efficient FANCD2 monoubiquitination. 122 FANCL was also shown to bind to PCNA directly and PCNA K164 monoubiquitination was shown to be required for FANCL chromatin loading.…”