Interplay between Fanconi anemia and homologous recombination pathways in genome integrity

Abstract

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error-free pathway for double-strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication-associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.

Keywords: DNA damage response; DNA repair; Fanconi anemia; genome stability; homologous recombination; replication stress.

Figure 1. Interstrand crosslink (ICL) and double strand break (DSB) repair pathways

(A) The Fanconi anemia (FA) pathway of ICL repair. Upon fork stalling at ICL sites, BRCA1 acts to dismantle the replisome (not shown) and RAD51 binds to the single‐stranded DNA to protect the fork. Subsequent FANCM–FAAP24–MHF1/2 complex binding activates ATR signalling and promotes recruitment of the FA core complex. The core complex in turn ubiquitinates the FANCI–FANCD2 heterodimer, which acts via SLX4 as a platform to recruit multiple nucleases (ERCC1‐XPF, SLX1 and MUS81‐EME). Nucleolytic incisions unhook the ICL and facilitate translesion synthesis‐dependent lesion bypass, mediated by REV1 or Polζ polymerases. The thus‐generated DSB is repaired by HR. (B) The HR pathway of DSB repair. DNA ends at a break site are resected to generate single‐stranded DNA tails. Resection is initiated by the MRN complex, stimulated though CtIP interaction and further extended though the activities of EXO1, BLM, WRN and DNA2. The resulting single‐stranded DNA is a substrate for RAD51 monomer loading in BRCA2‐ and RAD51 paralog‐dependent manner. The nucleoprotein filament thus generated invades a homologous double‐stranded DNA and, following second‐end capture, a double Holliday junction structure is generated. Branch migration facilitates cleavage of Holliday junctions by GEN1 or SLX4‐MUS81‐EME1‐SLX1 resolvases, or their dissolution dependent on the BLM–TOPIIIα–RMI1 complex. Crossover or non‐crossover molecules are the final products of the DNA repair reaction. Blue, FA proteins; red, HR proteins annotated as FA complementation groups; grey, other proteins associated with each pathway.

Figure 2. Fanconi anemia (FA) pathway activation in response to DNA damage and replication stress

(A) ICL‐induced fork stalling recruits the FANCM–FAAP24–MHF1/2 complex, which, in turn, activates ATR signalling. ATR phosphorylates components of the FA core complex (FANCA and FANCE) and FANCI–FANCD2. FA core complex recruitment to damage site leads to FANCI–FANCD2 monoubiquitination and chromatin binding to initiate repair. (B) IR‐induced DNA damage elicits ATM and ATR activation leading to phosphorylation of FANCD2. ATR is required for the efficient monoubiquitination of FANCD2 by the core complex, which triggers cell cycle arrest. (C) Low‐dose (e.g. 0.5 mM) HU treatment elicits ATR activation and FANCD2 binding to MCM2‐7, which limits DNA synthesis. Concomitantly, FANCI also binds the MCM complex to promote dormant origin firing. ATR‐dependent FANCI phosphorylation inhibits dormant origin firing and initiates DNA repair/replication fork restart. FANCD2 also inhibits FANCI‐mediated dormant origin firing, independently of its monoubiquitination status. (D) High‐dose (2–5 mM) HU treatment elicits activation of the classical FA pathway. BRCA1, BRCA2 and monoubiquitinated FANCD2 are recruited to stalled replication forks to protect them from degradation by stabilising RAD51 filaments on single‐stranded DNA.