Uvrabc Excision Of Interstrand Crosslink Mitomycin C-Dna Lesion Induces Double-Stranded Dna Breaks

Mitomycin C (MC) forms at least nine different covalent DNA adducts including an N2-2’-deoxyguanosine monoadduct (MC-mono-dG) and inter- and intrastrand bis-adducts with 2’-deoxyguanosine (dG) in vivo. Inter-strand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation, and therefore, the MC antitumor activity is most likely derived from formation of ICL-MC adducts. In Escherichia coli cells, UvrABC, the nucleotide excision repair (NER) enzyme complex can repair ICL psoralen lesions that cause DNA bending. The mechanism and the consequence of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. In order to compare the NER of MC-mono-dG with an ICL dG-MC-dG adduct, we constructed DNA fragments containing the adducts in the same DNA sequence context. We determined that UvrABC incises only at the strand containing MC-mono-dG adduct. In contrast, UvrABC makes three types of incisions on a site specific ICL dG-MC-dG adduct: type 1 (40 %), a single 5’ incision on one strand and 3’ incision on the other; type 2 (21 %), dual incisions on one strand and a single incision on the other strand; and type 3 (39%), dual incisions on both strand of both 5’ and 3’ sides of the ICL dG-MC-dG adduct. These three types of incisions occur with similar kinetics. An important consequence of these incisions is formation of DNA double-stranded breaks (DSB). Contrary to conventional model, we also found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically and form complexes that include UvrA. No UvrB- and UvrB+UvrCDNA complexes were observed. Our findings challenge the current UvrABC incision model of ICLs. We propose that NER of ICL dG-MC-dG adduct produces DSB, a lethal event that contributes to MC antitumor activity, and causes a non-homologous end-to-end joining that leads to DNA translocation and deletion mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1967.