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

N. V. Lukyanchikova1,2, I. O. Petruseva1, A. N. Evdokimov1, V. N. Silnikov1, and O. I. Lavrik1,2,3*

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
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 dCFAB and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dCFAB/dG (probe I), dCFAB/nFlu+4 (probe II), and dCFAB/nFlu–3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC–HR23B (Kdum > KdI > KdIIKdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dCFAB results in (i) decreased melting temperature (ΔTm = –3°C) and (ii) 12° DNA bending. The extended dCFAB/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 (dCFAB/nFlu+4, dCFAB/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.
KEY WORDS: DNA, bulky DNA lesions, protein factors of NER, photoaffinity labeling

DOI: 10.1134/S0006297916030093