Bipartite substrate discrimination by human nucleotide excision repair

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Abstract
Mammalian nucleotide excision repair (NER) eliminates carcinogen–DNA adducts by double endonucleolytic cleavage and subsequent release of 24–32 nucleotide-long single-stranded fragments. Here we manipulated the deoxyribose–phosphate backbone of DNA to analyze the mechanism by which damaged strands are discriminated as substrates for dual incision. We found that human NER is completely inactive on DNA duplexes containing single C4′-modified backbone residues. However, the same C4′ backbone variants, which by themselves do not perturb complementary hydrogen bonds, induced strong NER reactions when incorporated into short segments of mispaired bases. No oligonucleotide excision was detected when DNA contained abnormal base pairs without concomitant changes in deoxyribose–phosphate composition. Thus, neither C4′ backbone lesions nor improper base pairing stimulated human NER, but the combination of these two substrate alterations constituted an extremely potent signal for double DNA incision. In summary, we used C4′-modified backbone residues as molecular tools to dissect DNA damage recognition by human NER into separate components and identified a bipartite discrimination mechanism that requires changes in DNA chemistry with concurrent disruption of Watson–Crick base pairing.