Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Abstract

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for crosstalk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA monoubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

“…In response to bulky DNA lesions, Rad18-dependent FA pathway activation is mediated via PCNA ubiquitylation and requires Polη. 27 We show here that in contrast with bulky DNA lesions, CPT does not induce PCNA ubiquitination. Surprisingly, however, we demonstrate that Rad18-mediated FA pathway activation by CPT and tolerance of CPT-induced damage are dependent on Rad18 E3 ubiquitin ligase activity.…”

“…Indeed, deficiency or mutations in multiple components of the FA pathway (FANCB, FANCM, FANCJ, FANCD1, FANCD2 and FANCI) leads to CPT sensitivity. 36,[38][39][40] However, little is known regarding activation mechanisms of the FA pathway and its role in response to Top1-inhibitors-induced DNA damage.Because we and others recently showed that Rad18 contributes to FA pathway activation in response to bulky DNA adducts, 27 it was of interest to test the potential role (if any) of Rad18 in CPT-induced FA pathway activation. In response to bulky DNA lesions, Rad18-dependent FA pathway activation is mediated via PCNA ubiquitylation and requires Polη.…”

“…Because we and others recently showed that Rad18 contributes to FA pathway activation in response to bulky DNA adducts, 27 it was of interest to test the potential role (if any) of Rad18 in CPT-induced FA pathway activation. In response to bulky DNA lesions, Rad18-dependent FA pathway activation is mediated via PCNA ubiquitylation and requires Polη.…”

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.…”