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DNA nterstrand cross-links (ICLs) are cytotoxic products of common anticancer drugs and cellular metabolic processes, whose mechanism(s) of repair remains poorly understood. In this study, we show that cross-link structure affects ICL repair in nonreplicating reporter plasmids that contain a mispaired N4C-ethyl-N4C (C-C),. N3T-ethyl-N3T (T.-T), or. Nll-ethyl-N3T (l-T) ICL. The T-T and l-T cross-links obstruct the hydrogen bond face of the base and mimic the NIG-ethyl-N3C ICL created by bis-chloroethylnitrosourea, whereas the C-C cross-link does not interfere with base pair formation. Host-cell reactivation (HCR) assays in human and hamster cells showed that repair of these ICLs primarily involves the transcription-coupled nucleotide excision repair (TC-NER) pathway. Repair of the C-C ICL was 5-fold more efficient than repair of the T-T or I-T ICLs, suggesting the latter cross-links hinder lesion bypass following initial ICL unhooking. The level of luciferase expression from plasmids containing a C-C cross-link remnant on either the transcribed or nontranscribed strand increased in NER-deficient cells, indicating NER involvement occurs at a step prior to remnant removal, whereas expression from similar T-T remnant plasmids was inhibited in NER-deficient cells, demonstrating NER is required for remnant removal. Sequence analysis of repaired plasmids showed a high proportion of C residues inserted at the site of the T-T and l-T cross-links, and HCR assays showed that RevI was likely responsible for these insertions. In Contrast, both C and G residues were inserted at the C-C cross-link site, and Revl was not required for repair, suggesting replicative or other translesion polymerases can bypass the C-C remnant. [ABSTRACT FROM AUTHOR]

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 ICLinduced 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. [ABSTRACT FROM AUTHOR]

We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in living mammalian cells. We have described psoralen-linked TFOs with 2'-0-methyl and 2'-0-(2-aminoethyl) (2'-AE) substitutions that are active in a gene knockout assay in cultured cells. The assay is based on mutagenesis by psoralen, a photoactive DNA cross-linker. Previous work showed that TFOs with three or four 2'-AE residues were disproportionately more active than those with one or two substitutions. Here we demonstrate that for optimal bioactivity the 2'-AE residues must be clustered rather than dispersed. We have further characterized bioactive and inactive TFOs in an effort to identify biochemical and biophysical correlates of biological activity. While thermal stability is a standard monitor of TFO biophysical activity, we find that T[Subm] values do not distinguish bioactive and inactive TFOs. In contrast, measurements of TFO association rates appear to correlate well with bioactivity, in that triplex formation occurs disproportionately faster with the TFOs containing three or four 2'-AE residues. We asked if extending the incubation time prior to photoactivation would enhance the bioactivity of a TFO with a slow on rate relative to the TFO with a faster association rate. However, there was no change in bioactivity differential. These results are compatible with a model in which TFO binding in vivo is followed by relatively rapid elution by cellular functions, similar to that described for transcription factors. Under these circumstances, TFOs with faster on rates would be favored because they would be more likely to be in triplexes at the time of photoactivation. [ABSTRACT FROM AUTHOR]