Distortion-dependent unhooking of interstrand cross-links in mammalian cell extracts

Abstract

Interstrand cross-links (ICLs) are formed by many chemotherapeutic agents and may also arise endogenously. The mechanisms used to repair these lesions remain unclear in mammalian cells. Repair in Escherichia coli and Saccharomyces cerevisiae requires an initial unhooking step to release the tethered DNA strands. We used a panel of linear substrates containing different site-specific ICLs to characterize how structure affects ICL processing in mammalian cell extracts. We demonstrate that ICL-induced distortions affect NER-dependent and -independent processing events. The NER-dependent pathway produces dual incisions 5' to the site of the ICL as described previously [Bessho, T., et al. (1997) Mol. Cell. Biol. 17 (12), 6822-6830] but does not release the cross-link. Surprisingly, we also found that the interstrand cross-linked duplexes were unhooked in mammalian cell extracts in a manner independent of the NER pathway. Unhooking occurred identically in extracts prepared from human and rodent cells and is dependent on ATP hydrolysis and metal ions. The structure of the unhooked product was characterized and was found to contain the remnant of the cross-link. Both the NER-mediated dual 5' incisions and unhooking reactions were greatly stimulated by ICL-induced distortions, including increased local flexibility and disruption of base pairs surrounding the site of the ICL. These results suggest that in DNA not undergoing transcription or replication, distortions induced by the presence of an ICL could contribute significantly to initial cross-link recognition and processing.

Figure 1

Substrates used in this study. (A) Schematic of ICLs where the top left end of each duplex represents the 5’ end. (B) Sequences of cross-linked duplexes used in the incision assays. (C) Sequence of full length substrate where [X] denotes the cross-link duplex from (B) ligated in the middle. Bold italic nucleotides contain 3’ methylphosphonate linkages. = ³²P label positions 1-5; ■ denotes every 10^th base from the 5’ end; ▼ denotes every 10^th base from the 3’ side;

Figure 2

Chemical probing of -CG- and -GC- ICLs. Arrows indicate bases susceptible to chemical probes due to structural distortions. K=KMnO₄; D=DMS + piperidine; P=piperidine only; C= control non-cross-link.

Figure 3

Processing of ICL substrates on the 5’-side of the cross-link in mammalian cell extracts. ICL substrates were labeled with ³²P on the 5’ side of the (A) -CG- and -GC- (at positions 1 and 2) or (B) psoralen ICL (position 1) and incubated with wild-type human or CHO (HeLa or AA8, respectively), NER deficient UV41 (XPF), UV135 (XPG) or a mixture of XPF and XPG (F+G) extracts and then processed and analyzed by denaturing PAGE as described in Materials and Methods. * and ** = single 5’ incision; NER = dual 5’ incisions made by the nucleotide excision repair pathway; M = molecular weight markers; ND = non damaged substrate with same sequence as the psoralen ICL duplex. (C) -GC- ICL was incubated with AA8 WCE with the indicated modifications to the standard reaction conditions. (D) Schematic illustrating the single 5’ incision locations on -CG-, -GC- and AMT ICLs (mapped in Figure S2A). The dual NER incision locations were not determined here but were previously for psoralen (10). = ³²P label; arrows indicate location of incisions.

Figure 4

Processing of ICL substrates on the 3’-side of the cross-link in mammalian cell extracts. ICL substrates were labeled with ³²P on the 3’ side at position 3 of the (A) -CG- and -GC- or (B) psoralen ICL, incubated with wild type or NER deficient UV41 (XPF), UV135 (XPG) or a mixture of XPF and XPG (F+G) extracts and then processed and analyzed by denaturing PAGE as described in Materials and Methods. (C) The -GC- ICL was incubated with AA8 WCE under the indicated modifications to the standard reaction. (D) Schematic illustrating exact location of 3’ incisions on -CG-, -GC- and AMT ICLs (mapped in Figure S1B). = ³²P label; arrows indicate location of incisions.

Figure 5

Unhooking of ICL substrates in mammalian cell extracts. (A) ³²P labeled ICL substrates were incubated with the indicated WCE and then processed and analyzed on a 6% denaturing gel run at 60°C. (B) Requirements for unhooking reaction. -GC- ICL was incubated with AA8 WCE with the indicated modifications to the standard reaction conditions. The percent unhooked product was determined and is indicated beneath the gel. Quantifications of the unhooking reactions in Panel (A) are shown in Figure 7. (C) Schematic of possible structures of product observed in (A) and (B). (D) Characterization of unhooked product. The unhooked product was gel purified and run on an 8% sequencing gel next to a non-damaged single stranded control. Half of the purified unhooked product was subjected to reversal of the ICL: GC by sodium bisulfite treatment and psoralen ICL by short wavelength UV. Loss of some sample due to the handling of the reversal treatment resulted in less DNA loaded in the treated lanes. C = non-damaged control.

Figure 6

Unhooking is independent of NER. Cross-linked substrates were incubated with the indicated NER deficient extract and processed as described in Materials and Methods. XL_o denotes the migration position of the non-cross-linked control duplex.

Figure 7

Unhooking is influenced by ICL-induced DNA distortions. (A) 3-5 reactions with the indicated ICL were carried out in AA8 WCE for 60 min and then processed as described in Materials and Methods. The unhooked product was quantified relative to the total counts loaded. Error bars represent the standard error from the mean.

Figure 8

Schematic illustrating NER-dependent and independent processing of ICLs observed in mammalian cell extracts. Both types of processing are sensitive to ICL induced helical distortions in latent DNA.