DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates

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N. V. Lukyanchikova^1,2, I. O. Petruseva¹, A. N. Evdokimov¹, V. N. Silnikov¹, and O. I. Lavrik^1,2,3*

¹Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia; E-mail: lavrik@niboch.nsc.ru

²Novosibirsk State University, 630090 Novosibirsk, Russia

³Altai State University, 656049 Barnaul, Russia

^* To whom correspondence should be addressed.

Received August 6, 2015; Revision received September 25, 2015

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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.

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KEY WORDS: DNA, bulky DNA lesions, protein factors of NER, photoaffinity labeling

DOI: 10.1134/S0006297916030093

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