Rad18 emerges as a critical regulator of the Fanconi anemia pathway

Fanconi anemia (FA) is a genetic disorder resulting in defective repair of DNA damage, genomic abnormalities and the development of cancer. The disease can be caused by defects in at least 13 different genes that function within the FA pathway and is characterized by hypersensitivity to DNA interstrand crosslinking agents such as cisplatin and mitomycin c.¹ Such DNA lesions cause stalling of DNA replication forks, resulting in the activation of the FA pathway and the monoubiquitination of FANCD2 by the FA core complex that contains an E3 ubiquitin ligase. The monoubiquitination of FANCD2 is required for FANCD2 association with chromatin, where it coordinates the repair of DNA damage by nucleotide excision repair, translesion synthesis and homologous recombination.¹

Recently, Vaziri and colleagues demonstrated that the FA pathway becomes activated in response to bulky DNA adducts induced by BPDE in a Rad18-dependent manner.² Rad18 is a conserved E3 ligase that monoubiquitinates the replication factor PCNA in response to stalled replication forks at UV-induced lesions. Monoubiquitination of PCNA recruits bypass polymerases that allow replication to pass the lesion. Song et al. demonstrated that PCNA monoubiquitination by Rad18 was necessary for the activation of FANCD2 monoubiquitination in response to BPDE.²

Given that both Rad18- and FA-deficient cells are also sensitive to replication-induced double-strand breaks mediated by the Topoisomerase I inhibitor camptothecin (CPT), Palle and Vaziri questioned whether Rad18 was also playing a role in activation of the FA pathway in response to CPT.³ The authors showed that low doses of CPT induce FANCD2 ubiquitination, chromatin loading and assembly into foci. Depletion or absence of Rad18 dramatically reduced FANCD2 monoubiquitination and chromatin loading, both basally and after CPT treatment. This reduction in FANCD2 recruitment is functionally important, as depletion of FANCD2 or Rad18 resulted in hypersensitivity to CPT, persistent DNA damage and reduced recovery of DNA replication.

The authors go on to demonstrate that while Rad18's ubiquitin ligase activity is required to promote chromatin loading of FANCD2, PCNA monoubiquitination is not detected after treatment with CPT. These observations are similar to a recent study that reported that Rad18's ubiquitin ligase activity is required independently of PCNA monoubiquitination for the efficient monoubiquitination of FANCD2 in response to mitomycin c.⁴ Thus, Rad18 can promote FANCD2 ubiquitination by a PCNA-dependent mechanism in response to bulky DNA adducts² and a PCNA-independent mechanism in response to interstrand crosslinks⁴ and replication-induced double-strand breaks.³

Why is Rad18 required for efficient FANCD2 monoubiquitination? It is unlikely that Rad18 is directly ubiquitinating FANCD2, as it is well established that the FA core complex is required for FACND2 ubiquitination, and there is no evidence that FANCD2 is a RAD18 substrate.^2,4 The FANCD2 deubiquitinase Usp1 has been demonstrated to be downregulated in response to DNA damage.^5,6 The authors, however, found no evidence of CPT-induced or Rad18-mediated change in Usp1 levels and thus conclude that Rad18 is not regulating FANCD2 deubiquitination.³ Instead, Palle and Vaziri suggest that Rad18 may be necessary for FA core complex recruitment to and/or retention on chromatin.

In conclusion, Rad18 has a critical role in response to replication-fork stalling in the presence of various types of DNA damage by a number of mechanisms. In response to UV and bulky adducts, Rad18 monoubiquitinates PCNA, which then recruits bypass polymerases to allow DNA synthesis past the lesion. This leads to activation of the FA pathway and to chromatin loading of monoubiquitinated FANCD2.² In contrast, when a replication fork stalls due to an interstrand crosslink or a toposiomerase I cleavage complex, Rad18's ubiquitin ligase activity is required for proper activation of the FA pathway via an unknown mechanism.^3,4 Lastly, Rad18 stimulates homologous recombination in an ubiquitin ligaseindependent manner by interacting with and recruiting Rad51C to double-strand breaks.⁵

This work raises several interesting questions to be addressed. First, given that Rad18's role in the FA pathway is not dependent on PCNA, is there an unidentified Rad18 substrate that regulates FANCD2 monoubiquitination? Possible mechanisms could include activating ubiquitination of FA core complex subunits or inhibiting the catalytic activity of the Usp1 deubiqutinase. Second, are there other E3 ubiquitin ligases involved in activation of the FA pathway? One such possibility is the CRL4^Cdt2 complex which also monoubiquitinates PCNA⁷ and thus may recognize other Rad18 substrates. Next, is Rad18 regulating both basal and damage-induced monoubiquitination of FANCD2 levels by the same mechanism? Additionally, is Rad18 involved in FANCD2 monoubiquitination in response to replication stress induced by anticancer drugs such as hydroxyurea or inducers of re-replication such as MLN4924? Finally, if Rad18 is important for activating the Fanconi pathway in the presence of specific types of DNA damage, are there naturally occurring somatic or acquired mutations in Rad18 that give rise to diseases with phenotypes like Fanconi anemia? Furthermore, can mutations in Rad18 be found that affect the FA pathway without affecting Rad18's ability to monoubiquitinate PCNA?