The Fanconi Anemia Pathway Promotes Replication-Dependent DNA Interstrand Cross-Link Repair

Abstract

Fanconi anemia is a human cancer predisposition syndrome caused by mutations in thirteen Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand crosslinks (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. We make use of a cell-free system to show that the FANCI-FANCD2 complex is required for replication-dependent ICL repair. Removal of FANCD2 from extracts inhibits nucleolytic incisions near the ICL as well as translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.

Recruitment and positioning determine the specific role of the XPF ‐ ERCC 1 endonuclease in interstrand crosslink repair

“…Replication intermediates were linearized and separated on a denaturing agarose gel. At early times, the parental strand migrates as a large X‐structure, while after crosslink unhooking during repair, it is converted to a linear molecule and arms (Fig 4A; Knipscheer et al , 2009). The decline of the X‐shaped structures and the accumulation of the linears are a direct readout of unhooking incisions.…”

“…Dual incisions on either side of the ICL then unhook the lesion from one of the strands. This critical repair step requires the endonuclease XPF (FANCQ)‐ERCC1, which is recruited to the ICL by the large scaffold protein SLX4 (FANCP), and depends on the activation of the Fanconi anemia pathway by ubiquitylation of the FANCI‐FANCD2 complex (Knipscheer et al , 2009; Klein Douwel et al , 2014). After unhooking, a nucleotide is inserted across from the adducted base, followed by strand extension by REV1 and polymerase ζ, consisting of REV7 (FANCV) and REV3 (Räschle et al , 2008; Budzowska et al , 2015; Mamrak et al , 2016).…”

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

“…[42][43][44] A functional FA pathway is required for efficient incision of bidirectionally arrested replication forks at an interstrand DNA crosslink in frog egg extracts. 32,45 It will be informative to study the impact of FA pathway dysfunction on Tus/Ter-induced HR in mammalian cells.…”

Fanconi anemia: a model disease for studies on human genetics and advanced therapeutics

“…The activated FANCore complex monoubiquitinates the FANCD2-FANCI heterodimer (ID complex) [36,37] and the activated ID complex relocates to the damaged DNA in an ATR and BRCA1-dependent manner [36][37][38]. The ID complex promotes nucleolytic cleavage of the 3´ and 5´ sites of DNA to unhook the ICL and successively induces trans-lesion polymerases Rev1 and pol [39][40][41][42]. These reactions extend the leading DNA strand above and past the unhooked ICL to produce a substrate that is processed by successive HRR reactions [34].…”