Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication

Abstract

Homologous recombination (also termed homology-directed repair, HDR) is a major pathway for the repair of DNA interstrand cross-links (ICLs) in mammalian cells. Cells from individuals with Fanconi anemia (FA) are characterized by extreme ICL sensitivity, but their reported defect in HDR is mild. Here we examined ICL-induced HDR using a GFP reporter and observed a profound defect in ICL-induced HDR in FA cells, but only when the reporter could replicate.

Figure 1. ICL induction of HDR in mammalian cells

(a) HDR reporters. After DNA damage in the upstream GFP repeat, HDR with the downstream GFP repeat leads to a GFP⁺ gene. A site-specific ICL forms from psoralen conjugated to a TFO (pso-TFO). A site-specific DSB is formed by I-SceI endonuclease. (b) ICL formation in TR-GFP. The TFO binds to the TR-GFP plasmid sequence forming a triplex. Psoralen intercalates at the TpA site, which after UVA exposure forms an ICL, blocking DraI cleavage. Lower case nucleotides within the TFO are locked nucleic acids (LNA) which enhance the stability of the triplex in vivo; all cytosines are methylated at position 5. (c) DraI protection assay to quantify in vivo ICL formation. DNA was isolated from U2OS cells transfected with pso-TFO:TR-GFP and treated with UVA. Unconjugated TFO was the control in the left panel and a conjugated TFO which cannot bind TR-GFP (pso-mTFO) was the control in the right panel. ICL formation is distinguished from non-covalent triplex DNA formation by addition of an oligonucleotide complementary to the TFO prior to DraI cleavage which binds to the TFO released from the triplex upon heating (Supplementary Fig. 1d). The 3.5-kb fragment is indicative of ICL formation (arrow). Thin bar, probe. (d) ICL-induced HDR in U2OS cells. Triplexes formed in vitro were transfected into U2OS cells which were then treated with UVA. GFP⁺ cells arise from HDR of the ICL in TR-GFP, and are significantly lower without UVA or pso-TFO (p=0.0003, unpaired t test). Transfection efficiency is monitored by cotransfection of a DsRed expression vector. Error bars, ±1 SD; n=4 replicates from 2 independent experiments. (e) ICL-induced HDR, like DSB-induced HDR, is significantly reduced in Brca2 mutant V-C8 cells compared with BRCA2-complemented V-C8 cells (p<0.0001). Triplexes formed between TR-GFP and either pso-TFO (pso) or an unmodified TFO (+) were transfected into cells which were then treated with UVA. For DSB-induced HDR, DR-GFP was transfected into cells with or without the I-SceI expression vector. n=5 replicates from 2 independent experiments.

Figure 2. ICL and DSB-induced HDR in mouse ES cell mutants

Percentage GFP⁺ cells for each mutant was normalized to the indicated control. BRCA1-deficient cells have substantially reduced ICL and DSB-induced HDR (a); Ku70-deficient cells only have increased DSB-induced HDR (b); ERCC1-deficient cells only have reduced ICL-induced HDR (c); FANCA-deficient cells have mildly reduced ICL and DSB-induced HDR (d). For ICL-induced HDR, cells were transfected with pso-TFO:TR-GFP or TFO:TR-GFP and then treated with UVA. Error bars, ±1 SD. See Supplementary Table 1 for statistical analysis.

Figure 3. FANCA-dependent, replication-coupled ICL-induced HDR

(a) HDR reporters with EBV OriP for replication in EBNA1-expressing cells. (b-e) HDR assays in U2OS cells stably expressing EBNA1 (+) or not (–). DSB-induced HDR is only moderately increased with EBNA1/OriP (b), whereas ICL-induced HDR is substantially increased with EBNA1/OriP from either in vivo (c) or in vitro ICL formation without (d) or with (e) removal of the TFO tail. All comparisons of I-SceI or pso-TFO plus or minus EBNA1/OriP have p≤0.0002. Error bars, ±1 SD; n=4 or 5 replicates from 2 independent experiments. (f-i). HDR assays in GM6914 or FANCA-complemented GM6914 cells expressing EBNA1 (+) or not (–). DSB-induced HDR is moderately increased with FANCA and EBNA1/OriP (f). With EBNA1, ±FANCA p=0.0001; without EBNA1, ±FANCA p=0.005; with FANCA, ±EBNA1 p=0.004; without FANCA, ±EBNA1 p=0.26. n=4 replicates from 2 independent experiments. By contrast, ICL-induced HDR is substantially increased with FANCA and EBNA1/OriP from either in vivo (g) or in vitro ICL formation without (h) or with (i) removal of the TFO tail. With EBNA1 and FANCA, p<0.0001 compared with any other condition. Error bars, ±1 SD; n=4 to 6 replicates from 2 or 3 independent experiments (g,h); n=3 replicates from 1 experiment (i). For in vivo ICL formation, TR-OriP-GFP bound to the indicated triplex was transfected into cells which were then treated with UVA. For in vitro TFO formation, TR-OriP-GFP was bound to the indicated triplex and then exposed to UVA prior to transfection. j. Model for ICL repair by HDR. ICLs stall converging replication forks. Incisions on both sides of the ICL create strand breaks, and translesion synthesis (TLS) allows replication past the lesion. The adduct is removed and strand invasion leads to HDR. As replication from OriP has been reported to be predominately unidirectional, HDR in TR-GFP likely proceeds after stalling of a single replication fork.