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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]