DNA interstrand crosslink repair and cancer

Abstract

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that prevent transcription and replication by inhibiting DNA strand separation. Agents that induce ICLs were one of the earliest, and are still the most widely used, forms of chemotherapeutic drug. Only recently, however, have we begun to understand how cells repair these lesions. Important insights have come from studies of individuals with Fanconi anaemia (FA), a rare genetic disorder that leads to ICL sensitivity. Understanding how the FA pathway links nucleases, helicases and other DNA-processing enzymes should lead to more targeted uses of ICL-inducing agents in cancer treatment and could provide novel insights into drug resistance.

Figure 1. How ICLs kill tumour cells

Interstrand crosslinks (ICLs) can block the progression of the replication fork by inhibiting the progression of the replisome. ICLs can stall transcription. ICLs may distort the structure of chromatin and prevent the access of DNA-interacting proteins. Tumour cell death can be induced by p53- and FAS ligand-dependent apoptosis (programmed cell death) or p53-independent mitotic catastrophe (death when apoptosis is absent but vital DNA integrity is lost).

Figure 2. Activation of the Fanconi anaemia pathway coordinates DNA repair at an ICL

Interstrand crosslink (ICL) recognition by FANCM and associated proteins leads to the recruitment of the Fanconi anaemia (FA) core complex and monoubiquitylation (Ub) of FANCD2–FANCI. Retention of FANCD2–FANCI in the chromatin is followed by the recruitment of nucleases and polymerases that are required for the repair process. FANCM also recruits the Bloom’s syndrome complex (BTR), and is involved in activating cell cycle checkpoints through the replication protein A (RPA)–ataxia telangiectasia and Rad3-related (ATR)–CHK1 signalling cascade. The BRCA1 and BRCA2 complexes initiate and regulate homologous recombination leading to the stabilization of the stalled replication fork. FAN1, Fanconi-associated nuclease 1; Pol ν, DNA polymerase ν; RMI, RecQ-mediated genome instability; TOPOIIIα, topoisomerase IIIα.

Figure 3. ICL repair at different stages of the cell cycle

In cells in G1 phase of the cell cycle, nucleotide excision repair (NER) can remove a subset of interstrand crosslinks (ICLs). Excision by XPF–ERCC1 is followed by translesion synthesis, and excision of the ‘flipped out’ ICL. However, some lesions cannot be bypassed in this manner and a futile cycle of repair involving excision and ligation occurs. During S phase, a partially processed intermediate may be encountered by a replication fork, which leads to the collapse of the replication fork and the formation of a one-ended double-strand break (DSB). These collapses are probably avoided by FANCM-mediated fork regression and stabilization of the fork by the Fanconi anaemia (FA) pathway. This FA-stabilized homologous recombination (HR) intermediate (highlighted by beige circles) allows the nascent leading strand of DNA synthesis (indicated by a green arrow) to be extended by translesion synthesis polymerases (Pol; black dashed arrow). Unhooking of the ICL by coordinated incision by the 5′- and the 3′-flap endonucleases allows extension past the lesion. HR is then completed and replication can be re-established. If a second replication fork has encountered the ICL from the opposite direction, unhooking on the 3′ side cuts the leading strand template. The extension of the ‘left-hand’ leading strand would lead to its joining with the lagging strand of the right-hand fork, followed by second end capture and extension of the right-hand leading strand and the left-hand template strand. This process would complete replication without the re-initiation of lagging strand synthesis but would generate a double Holliday junction. This structure is resolved by the BLM-containing complex or may in some instances be cut by structure-specific nucleases (not shown). FAN1, Fanconi-associated nuclease 1.

Figure 4. Suppression of ICL sensitivity by inhibition of non-homologous end Nature Reviews Cancer joining

In wild-type cells, interstrand crosslinks (ICLs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The Fanconi anaemia (FA) pathway, however, promotes HR-dependent stabilization of the replication fork and DNA repair. In FA cells, double-strand breaks (DSBs) are frequently created that can be repaired by either HR or NHEJ. Because the DSB is one ended, NHEJ does not have a natural substrate to rejoin so these breaks can remain unrepaired (generating chromatid breaks) or may ligate with a DSB in a different chromosome (generating radial chromosomes). Suppression of NHEJ by deleting a component of this pathway or by using DNA-dependent protein kinase catalytic subunit (DNA-PKcs) inhibitors promotes the repair of the DSBs by FA-independent pathways of HR (dashed arrow).