Checkpoint signaling from a single DNA interstrand crosslink

Abstract

DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited, because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from, and repairing of a single site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivity to crosslinking agents results from both checkpoint and DNA repair defects.

Figure 1. Replication of ICLs in Xenopus cell-free extract

(A) Schematic representation of site-specific ICL and X and C primers. (B) PCR products of control and ICL plasmids with X and C primers. (C) qRT-PCR quantification of ratio of products across X and C primers (X:C ratio) for control and ICL plasmids. Mean ± SEM. (D) Electrophoretic analysis of nicked (N), linear (L), and supercoiled (SC) ICL plasmid (left panel). Electrophoretic analysis of replication and repair of control (center panel) and ICL plasmids (right panel) in the Xenopus HSS/NPE system in the presence of ³²P-αdCTP (CC: concatemer, SC: supercoiled, * replication/repair intermediates).

Figure 2. Replication initiation-dependent and -independent repair of ICLs

(A) PCR products using X and C primers on ICL-plasmid incubated in HSS/NPE for times indicated. (B) qRT-PCR quantification of X:C ratio for ICL-plasmid incubated in HSS/NPE for times indicated. (C) qRT-PCR quantification of X:C ratio of ICL-plasmid incubated for 75 min in non-treated (NT) extracts or extracts treated 50 ng/μL geminin and 500 μM roscovitine (GR). (D) HeLa cells were arrested by double thymidine block and transfected with ICL plasmid then released (NT) or maintained in thymidine block (Thymidine). 24 hr post transfection plasmid DNA was recovered for qRT-PCR quantification of X:C ratio. (E) Control and ICL plasmids were incubated in HSS/NPE for 0 min (○ Control, □ ICL) or 120 min (● Control, ■ ICL) in the presence of 500 mM BrdUTP and geminin/roscovitine. DNA was separated on a 1.72 g/mL CsCl gradient and analyzed by qRT-PCR using C primers for total DNA content. (F) ICL plasmid was replicated in HSS/NPE for 150 min in the absence or presence of geminin/roscovitine and amplified with A and B primers. PCR products were cloned and sequenced. (G) qRT-PCR quantification of X:C ratio in mock- and FANCL-depleted HSS/NPE (grey bars), in mock- and FANCL-depleted HSS/NPE with geminin and roscovitine (striped bars), or in non-treated (NT) or curcumin-treated (curcumin) HSS/NPE (black bars) for 90 min. Values normalized to the mock or NT controls. (H) PD20, PD20 + wtFANCD2, GM6914, GM6914 + wtFANCA were transfected with ICL-plasmid. 24 hr post transfection plasmid DNA was recovered and subjected to qRT-PCR quantification of X:C ratio.

Figure 3. ICLs activate a FA- and ATR-Chk1-dependent checkpoint

(A) Electrophoretic analysis of control or ICL plasmid with or without 5 mM caffeine incubated in HSS/NPE for 120 min, in the presence of ³²P-αdCTP. (B) 2.7 kb control or ICL plasmids were incubated in HSS/NPE for 90 min followed by the addition of a 3 kb plasmid (dashed) and ³²P-αdCTP for 20 min. DNA was analyzed on agarose gel. (C) HSS/NPE extracts in the presence of buffer (NT), 5 mM caffeine (Caffeine), or geminin and roscovitine (GR) were incubated with control (left panel) or ICL plasmid (right panel) for 90 min. Western blot analysis of soluble extracts was performed with the indicated antibodies. (D) ICL plasmid was incubated in HSS/NPE with or without 100 μM curcumin for 90 min, in the presence of ³²P-αdCTP. Nucleotide incorporation was quantified using phosphoimager and SEM is shown for 4 independent experiments. (E) 2.7 kb control or ICL plasmids were incubated in HSS/NPE with buffer (NT), 100 μM curcumin or 5 mM caffeine for 90 min, followed by the addition of a 3 kb plasmid (dashed) and ³²P-αdCTP for 20 min. DNA was analyzed on agarose gel.

Figure 4. ICL-checkpoint requires the FA pathway upstream of Chk1 activation

(A) Western blot analysis of mock- FANCD2-, and FANCD2-depleted HSS/NPE extracts supplemented with wt GST-xFANCD2 HSS/NPE extracts following incubation with ICL plasmid. Soluble extract was probed with indicated antibodies. (B) HSS/NPE extracts were incubated with ICL plasmid in buffer (NT) or 100 μM Curcumin. Soluble extract was probed with indicated antibodies. (C) HSS/NPE extracts were incubated with ICL plasmid in buffer (NT) or 5 mM DDN. Soluble extract was probed with indicated antibodies. (D) Western blot analysis of mock- and FANCL-depleted HSS/NPE extracts incubated with ICL plasmid. Soluble extract was probed with indicated antibodies. (E) Western blot analysis of mock- and FANCL-depleted HSS/NPE extracts incubated with ICL plasmid. DNA-bound proteins were probed with indicated antibodies. (F) Western blot analysis of mock- and FANCL-depleted HSS/NPE extracts in the presence of geminin and roscovitine incubated with ICL plasmid. Soluble extract was probed with indicated antibodies. (G) Western blot analysis of mock- and FANCL-depleted HSS/NPE extracts in the presence of geminin and roscovitine incubated with ICL plasmid. DNA-bound proteins were probed with indicated antibodies. For soluble extracts, MCM6 was used as a loading control and for DNA-bound proteins were normalized by qRT-PCR quantification of DNA for each sample (Table S3). ICL-plasmid was incubated in extracts for 90 min.

Figure 5. Mechanisms of ICL-induced checkpoint

(A) Western blot analysis of mock- and RPA-depleted HSS/NPE extracts incubated with ICL plasmid. Soluble extract was probed with indicated antibodies. (B) Western blot analysis of mock- and RPA-depleted HSS/NPE extracts incubated with ICL plasmid. DNA-bound proteins were probed with indicated antibodies. (C) Western blot analysis of mock- and ATR-depleted HSS/NPE extracts incubated with ICL plasmid. Soluble extract was probed with indicated antibodies. (D) Western blot analysis of mock- and ATR-depleted HSS/NPE extracts incubated with ICL plasmid. DNA-bound proteins were probed with indicated antibodies. (E) Western blot analysis of mock- and ATR-depleted HSS/NPE extracts incubated with plasmids irradiated with 500 mJ/cm² UVB. (F) As in E, with mock- and FANCL-depleted extracts. For soluble extracts, MCM6 was used as a loading control and DNA-bound proteins were normalized by qRT-PCR quantification of DNA for each sample. Extracts were incubated with or without geminin and roscovitine as indicated. ICL or UV plasmids were incubated in extracts for 90 min.