Cross-link structure affects replication-independent DNA interstrand cross-link repair in mammalian cells.
DNA interstrand 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 N(4)C-ethyl-N(4)C (C-C), N3T-ethyl-N3T (T-T), or N1I-ethyl-N3T (I-T) ICL. The T-T and I-T cross-links obstruct the hydrogen bond face of the base and mimic the N1G-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 I-T cross-links, and HCR assays showed that Rev1 was likely responsible for these insertions. In contrast, both C and G residues were inserted at the C-C cross-link site, and Rev1 was not required for repair, suggesting replicative or other translesion polymerases can bypass the C-C remnant.
Hlavin
Erica M
EM
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.
Smeaton
Michael B
MB
Noronha
Anne M
AM
Wilds
Christopher J
CJ
Miller
Paul S
PS
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols; D006224:Cricetinae; D003520:Cyclophosphamide; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004317:Doxorubicin; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D014750:Vincristine
D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide; D004247:DNA