FANCD2-Controlled Chromatin Access of the Fanconi-Associated Nuclease FAN1 Is Crucial for the Recovery of Stalled Replication Forks

Abstract

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2(Ub)) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2(Ub) isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation.

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

“…After HU and APH treatment, Rad51 loaders, such as BRCA2, prevent Mre11-dependent nucleolytic degradation of nascent DNA at stalled replication forks (6,7,(9)(10)(11)(12)(13)(14)38). We therefore treated Rad51-depleted and control samples with mirin, a specific inhibitor of the exonuclease activity of Mre11 (39).…”

“…Over the past few years, it has become evident that Rad51 escorts ongoing replication forks regardless of the presence of DSBs (3)(4)(5). Specifically, Rad51 protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation and facilitates replication fork restart when the replication-halting agent hydroxyurea (HU) or aphidicolin (APH) is removed (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Such novel functions of Rad51 require many HRR factors, including BCRA2, FANCD2 (Fanconi Anemia Complementation group protein D2), CtIP, BRCA1, and the WRN helicase, but are independent of HRR effectors, such as Rad54 (6,7).…”

Fan1 deficiency results in DNA interstrand cross-link repair defects, enhanced tissue karyomegaly, and organ dysfunction

“…In yeast, Fan1 is capable of conferring ICL repair activity in the absence of the UBZ domain (Fontebasso et al 2013). One potential role of the interaction between FAN1 and FANCD2/FANCI outside of ICL repair includes the processing of replication forks under normal or replicative stress conditions (Schlacher et al 2012;Chaudhury et al 2014;Chen et al 2015). Most recently, it has been shown that the UBZ domain of FAN1 is essential for the processing of replication forks and protection against chromosome instability after treatment with HU and MMC (Lachaud et al 2016).…”

BRCA2 functions: from DNA repair to replication fork stabilization

“…The current model proposes that DNA2 and MRE11 accumulate on different types of stalled forks depending on their structures and the moment at which they appear (Karanja et al 2014, Higgs et al 2015, Thangavel et al 2015. Nevertheless, the recent finding that the Fanconi-associated nuclease 1 (FAN1) is also implicated in replication fork recovery (Lachaud et al 2016, Chaudhury et al 2014 suggests that this model is much more complex than initially anticipated.…”