The E3 ubiquitin ligase RAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI

Abstract

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wildtype counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. IntroductionFanconi anemia (FA) is an autosomal or X-linked recessive disorder characterized by genomic instability, congenital abnormalities, and a predisposition to cancer and leukemia. To date, 15 genes have been identified that, when mutated, result in FA or an FA-like syndrome. On a cellular level, these mutations can be characterized by hypersensitivity to DNA cross-linking agents such as diepoxybutane or mitomycin C (MMC). Consequently, the proteins encoded by these genes are thought to function in a common pathway responsible for the repair of interstrand cross-links (ICLs). [1][2][3] ICLs are complex lesions that covalently link double-stranded DNA, preventing replication and ultimately resulting in a double-strand break during repair. For this reason, several DNA repair factors are thought to function alongside FA proteins, including those involved in homologous recombination, nucleotide excision repair, and translesion synthesis (TLS). 4 Eight of the 15 FA proteins (FA complementation group A [FANCA], FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM) comprise what is known as the FA core complex, and a complete and functional core complex is required for the monoubiquitylation of FANCD2 and FANCI after DNA damage or during the S phase. 5 FANCL, along with the E2 protein UBE2T, functions as the E3 ubiquitin ligase component of the core complex responsible for the monoubiquitylation of FANCD2 and FANCI. [6][7][8] Monoubiquitylated FANCD2 and FANCI are readily loaded onto chromatin, 9 where they colocalize in nuclear repair foci with FANCD1, FANCJ, and FANCN, as well as other DNA repair factors such as BRCA1 and RAD51 and the DNA replication processivity factor proliferating cell nuclear antigen (PCNA). [10][11][12][13] RAD18 is an E3 ubiquitin ligase best known for its role in the monoubiquitylation of PCNA in response to stalled replication forks. [14][15] Monoubiquitylation of PCNA on lysine-164 by RAD18 and its partner E2 enzyme, RAD6, triggers a mechanism known as polymerase switching. 16 The sliding clamp, PCNA, normally carries a replicative polymerase such as pol␦ along the DNA strand during replication. When PCNA encounters a lesion caused by various DNA-dama...

ATR–Chk1–APC/CCdh1-dependent stabilization of Cdc7–ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress

“…This interaction likely facilitates not only a Cdc7-ASK-mediated phosphorylation of RAD18 to prime the latter for interaction with Pol h but also the recruitment of the RAD18-Pol h complex to stalled replication forks. In addition, our finding that the N-terminal region of RAD18 containing the RING domain interacts with ASK is quite intriguing given that FANCD2 and Rad51C also interact with the RING domain of RAD18 (Huang et al 2009;Williams et al 2011). It is possible that a binding partner of RAD18 may determine the optimal DNA damage response pathway upon distinct genotoxic insults.…”

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

“…After washing extensively with PBST to remove unbound secondary antibody, the slides were for FANC status by immunoblotting as described in recent publications. 45,64,65 All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, streptomycin sulfate (100 μg/ml) and penicillin (100 U/ml), 5 mM L-glutamine, and cultures were incubated at 37°C in humidified chambers with 5% CO 2 .…”

“…In a recent study, we reported that FANCD2 and Rad18 may be co-immunoprecipitated as a complex. 45 Whether Rad18 and FANCD2 associate directly or via other proteins is not known and is beyond the focus of this study. However, it is very common for DNA repair proteins to stabilize their binding partners, and this provides a plausible explanation for the effect of FANCD2-depletion on Rad18 levels.…”

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.…”