DNA with damage in both strands as affinity probes and nucleotide excision repair substrates

Resumo

Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC^FAB and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC^FAB/dG (probe I), dC^FAB/nFlu₊₄ (probe II), and dC^FAB/nFlu₋₃ (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC—HR23B (Kd_um > Kd_I > Kd_II ≈ Kd_III) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC^FAB results in (i) decreased melting temperature (ΔT_m = −3°C) and (ii) 12° DNA bending. The extended dC^FAB/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC—HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC^FAB/nFlu₊₄, dC^FAB/nFlu₋₃) or irreparable (nFlu/nFlu₊₄, nFlu/nFlu₋₃, nAnt/nFlu₊₄, nAnt/nFlu₋₃) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis.