Nucleotide Excision Repair of a DNA Interstrand Cross-Link Produces Single- and Double-Strand Breaks

Abstract

The DNA radical resulting from formal abstraction of a hydrogen atom from the thymidine methyl group, 5-(2'-deoxyuridinyl)methyl radical, forms interstrand cross-links with the opposing 2'-deoxyadenosine. This is the first chemically characterized, radical-mediated cross-link between two opposing nucleotides. In addition, cross-linking between opposing bases in the duplex is less common than between those separated by one or two nucleotides. The first step in cross-link repair was investigated using the UvrABC bacterial nucleotide excision repair system. UvrABC incised both strands of the cross-linked DNA, although the strand containing the cross-linked purine was preferred by the enzyme in two different duplexes. The incision sites in one strand were spaced 11-14 nucleotides apart, as is typical for UvrABC incision. The majority of incisions occur at the third phosphate from the 3'-side of the cross-link and eighth or ninth phosphate on the 5'-side. In addition, cleavage was found to occur on both strands, producing double-strand breaks in approximately 25-29% of the incision events. This is the first example of double-strand cleavage during nucleotide excision repair of cross-linked DNA that does not already contain a strand break in the vicinity of the cross-link.