Repair of cisplatin-induced DNA interstrand crosslinks by a replication-independent pathway involving transcription-coupled repair and translesion synthesis

Abstract

DNA interstrand crosslinks (ICLs) formed by antitumor agents, such as cisplatin or mitomycin C, are highly cytotoxic DNA lesions. Their repair is believed to be triggered primarily by the stalling of replication forks at ICLs in S-phase. There is, however, increasing evidence that ICL repair can also occur independently of replication. Using a reporter assay, we describe a pathway for the repair of cisplatin ICLs that depends on transcription-coupled nucleotide excision repair protein CSB, the general nucleotide excision repair factors XPA, XPF and XPG, but not the global genome nucleotide excision repair factor XPC. In this pathway, Rev1 and Polζ are involved in the error-free bypass of cisplatin ICLs. The requirement for CSB, Rev1 or Polζ is specific for the repair of ICLs, as the repair of cisplatin intrastrand crosslinks does not require these genes under identical conditions. We directly show that this pathway contributes to the removal of ICLs outside of S-phase. Finally, our studies reveal that defects in replication- and transcription-dependent pathways are additive in terms of cellular sensitivity to treatment with cisplatin or mitomycin C. We conclude that transcription- and replication-dependent pathways contribute to cellular survival following treatment with crosslinking agents.

Figure 1.

Construction of the plasmid containing site-specific cisplatin crosslinks. (A) pCX-RLuc plasmid with two BbsI sites cloned between the splice acceptor and the Renilla luciferase (hRluc) reporter gene. (B) The sequence of the cisplatin ICL oligonucleotide with the crosslinked guanine bases shown in bold. The ICL is located within a BsrBI restriction site. (C) The sequence of 1,3-GTG cisplatin intrastrand crosslink located within an ApaLI restriction site. The modified bases are shown in bold. (D) The sequence of 1,2-GG cisplatin intrastrand crosslink (modified bases in bold). (E) Analysis of the cisplatin ICL containing substrate. A 40-bp fragment was released from the plasmid by SacI digestion and labelled with α-[³²P]-dCTP. The fragments released from the cisplatin ICL (lane 1) or unmodified control plasmid (lane 2) were analysed by 12% denaturing PAGE. (F) Restriction analysis of the 1,3-GTG-cisplatin intrastrand crosslink substrate. ApaLI digest releases 3 fragments from unmodified control plasmid (1246, 2000 and 2055 bp, the last two are not separated in agarose gel) (lane 2). In the plasmid with the 1,3-GTG cisplatin intrastrand crosslink, the ApaLI site located at the position of the lesion is completely blocked (ApaLI digest yields fragments of 1246 and 4055 bp) (lane 1). Lane 3: 1 kb DNA Ladder (NEB).

Figure 2.

Reactivation of reporter gene expression blocked by a site-specific cisplatin ICL depends on NER. Plasmids containing site-specific cisplatin lesions: ICL (A) and 1,3-GTG- (B) or 1,2-GG- (C) intrastrand crosslink were transfected into normal human fibroblasts (WT, wild type) and fibroblasts from xeroderma pigmentosum patients groups A, F and G. XPA(+) are XP-A fibroblasts complemented with wild-type XPA. The relative repair is estimated as percentage of the reporter gene activity compared with the undamaged plasmid, after normalization to an internal cotransfected control. Standard deviations are shown as error bars.

Figure 3.

The repair of cisplatin ICLs depends on TC-NER, but not GG-NER. ICL- (A) or 1,3-GTG-intrastrand crosslink– (B) containing plasmids were transfected into XP-C (XPC), XP-C corrected with WT-XPC [XPC(+)], CS-B (CSB) and CS-B corrected with WT-CSB [CSB(+)] fibroblast cell lines and the relative repair activity measured as in Figure 2.

Figure 4.

The repair of cisplatin ICLs is defective in Rev1^−/− and Rev3^−/− cells, to a minor degree in Polκ^−/− cells, but not in XP-V and Polι^−/− cells. ICL- (A) or 1,3 GTG-intrastrand crosslink– (B) containing plasmids were transfected into WT, Rev3^−/− and Rev1^−/− mouse ES cells and the reactivation of luciferase gene expression measured. The same plasmids were transfected into Polη-deficient (XPV) and -complemented human XP-V [XPV(+)] cells and Polι- and Polκ-positive and deficient mouse ES cells, and levels of reporter gene expression determined (C, D).

Figure 5.

TC-NER–deficient, but not GG-NER deficient cells are sensitive to cisplatin, and the sensitivity is additive to FA pathway. XP-A (XPA) (A), XP-C (XPC) (B) and CS-B (CSB) (C) fibroblasts and the corresponding corrected cell lines [XPA(+), XPC(+), CSB(+)] were treated with the indicated doses of cisplatin and the surviving fraction determined using clonogenic assays. (D) CSB-deficient and -complemented cells were transfected with FANCD2-specific (siD2) and control (siLuc) siRNAs, treated with cisplatin and the surviving fraction determined using clonogenic assays. The data in A–D are plotted as the percentage of colonies that grew on the treated plates relative to untreated plates ± S.E. (error bars). (E) Western blot showing downregulation of FANCD2 in CS-B and corrected fibroblasts.

Figure 6.

Defective repair of MMC lesions in G1/G0 causes a stronger G2/M arrest in CSB-deficient cells versus corrected cells. CS-B (CSB) and complemented [CSB(+)] cells were arrested in G0/G1 by contact inhibition followed by 48 h serum starvation (0.1% FBS). MMC (10 ng/ml) was added or not added (NT) to the media for the last 16 h. Cells were further incubated in 0.1% FBS media without drug for 8 h, then trypsinized and released in complete media (time 0). Samples collected at times 0, 24 and 48 h were analysed by FACS after propidium iodide staining of the DNA.