Nucleotide Excision Repair of Chemically Stabilized Analogues of DNA Interstrand Cross-Links Produced from Oxidized Abasic Sites

Nucleotide excision repair is a primary pathway in cells for coping with DNA interstrand cross-links (ICLs). Recently, C4'-oxidized (C4-AP) and C5'-oxidized abasic sites (DOB) that are produced following hydrogen atom abstraction from the DNA backbone were found to produce ICLs. Because some of the ICLs derived from C4-AP and DOB are too unstable to characterize in biochemical processes, chemically stable analogues were synthesized [Ghosh, S., and Greenberg, M. M. (2014) J. Org. Chem. 79, 5948-5957]. UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB was examined. The incision pattern for the ICL related to the C4'-oxidized abasic site was typical for UvrABC substrates. UvrABC cleaved both strands of the substrate containing the C4-AP ICL analogue, but it was a poor substrate. UvrABC incised <30% of the C4-AP ICL analogue over an 8 h period, raising the possibility that this cross-link will be inefficiently repaired in cells. Furthermore, double-strand breaks were not detected upon incision of an internally labeled hairpin substrate containing the C4-AP ICL analogue. UvrABC incised the stabilized analogue of the DOB ICL more efficiently (similar to 20% in 1 h). Furthermore, the incision pattern was unique, and the cross-linked substrate was converted into a single product, a double-strand break. The template strand was exclusively incised on the template strand on the 3'-side of the cross-linked dA. Although the outcomes of the interaction between UvrABC and these two cross-linked substrates are different from one another, they provide additional examples of how seemingly simple lesions (C4-AP and DOB) can potentially exert significant deleterious effects on biochemical processes.