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A study was conducted to understand the cross-link structure of DNA interstrand that affects the repair in nonreplicating reporter plasmids that contain a mispaired [N.sup.4]C-ethyl-[N.sup.4]C (C-C), N3T-ethyl-N3T (T-T), or N1I-ethyl-N3T (I-T) ICL. The insertion of both, C and G residues at the C-C cross-link site without any need for Rev1 for repair indicated that replicative or other translesion polymerases could bypass the C-C remnant.

Interstrand cross-linked (ICL) linear DNA duplexes are used to examine cross-link processing in mammalian whole-cell extracts. The results have shown that in DNA not undergoing transcription or replication, distortions induced by the presence of an ICL has contributed to initial cross-link recognition and processing.

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]