Nucleotide excision repair (NER) efficiencies of DNA lesions can vary by orders of magnitude, for reasons that remain unclear. An example is the pair of N -(2′-deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N -(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) adducts that differ by a single acetyl group. The NER efficiencies in human HeLa cell extracts of these lesions are significantly different when placed at G ₁ , G ₂ or G ₃ in the duplex sequence (5′-CTCG ₁ G ₂ CG ₃ CCATC-3′) containing the Nar I mutational hot spot. Furthermore, the dG-C8-AAF adduct is a better substrate of NER than dG-C8-AF in all three Nar I sequence contexts. The conformations of each of these adducts were investigated by Molecular dynamics (MD) simulation methods. In the base-displaced conformational family, the greater repair susceptibility of dG-C8-AAF in all sequences stems from steric hindrance effects of the acetyl group which significantly diminish the adduct-base stabilizing van der Waals stacking interactions relative to the dG-C8-AF case. Base sequence context effects for each adduct are caused by differences in helix untwisting and minor groove opening that are derived from the differences in stacking patterns. Overall, the greater NER efficiencies are correlated with greater extents of base sequence-dependent local untwisting and minor groove opening together with weaker stacking interactions.

The hydroxyl radical is a powerful oxidant that generates DNA lesions including the stereoisomeric R and S 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) pairs that have been detected in cellular DNA. Unlike some other oxidatively generated DNA lesions, cdG and cdA are repaired by the human nucleotide excision repair (NER) apparatus. The relative NER efficiencies of all four cyclopurines were measured and compared in identical human HeLa cell extracts for the first time under identical conditions, using identical sequence contexts. The cdA and cdG lesions were excised with similar efficiencies, but the efficiencies for both 5′R cyclopurines were greater by a factor of ∼2 than for the 5′S lesions. Molecular modeling and dynamics simulations have revealed structural and energetic origins of this difference in NER-incision efficiencies. These lesions cause greater DNA backbone distortions and dynamics relative to unmodified DNA in 5′R than in 5′S stereoisomers, producing greater impairment in van der Waals stacking interaction energies in the 5′R cases. The locally impaired stacking interaction energies correlate with relative NER incision efficiencies, and explain these results on a structural basis in terms of differences in dynamic perturbations of the DNA backbone imposed by the R and S covalent 5′,8 bonds.

Failure in repairing ultraviolet radiation-induced DNA damage can lead to mutations and cancer. Among UV-lesions, the pyrimidine–pyrimidone (6-4) photoproduct (6-4PP) is removed from the genome much faster than the cyclobutane pyrimidine dimer (CPD), owing to the more efficient recognition of 6-4PP by XPC-RAD23B, a key initiator of global-genome nucleotide excision repair (NER). Here, we report a crystal structure of a Rad4–Rad23 (yeast XPC-Rad23B ortholog) bound to 6-4PP-containing DNA and 4-μs molecular dynamics (MD) simulations examining the initial binding of Rad4 to 6-4PP or CPD. This first structure of Rad4/XPC bound to a physiological substrate with matched DNA sequence shows that Rad4 flips out both 6-4PP-containing nucleotide pairs, forming an ‘open’ conformation. The MD trajectories detail how Rad4/XPC initiates ‘opening’ 6-4PP: Rad4 initially engages BHD2 to bend/untwist DNA from the minor groove, leading to unstacking and extrusion of the 6-4PP:AA nucleotide pairs towards the major groove. The 5′ partner adenine first flips out and is captured by a BHD2/3 groove, while the 3′ adenine extrudes episodically, facilitating ensuing insertion of the BHD3 β-hairpin to open DNA as in the crystal structure. However, CPD resists such Rad4-induced structural distortions. Untwisting/bending from the minor groove may be a common way to interrogate DNA in NER.

The molecular basis of resistance to nucleotide excision repair (NER) of certain bulky DNA lesions is poorly understood. To address this issue, we have studied NER in human HeLa cell extracts of two topologically distinct lesions, one derived from benzo[ a ]pyrene (10 R -(+)- cis-anti -B[ a ]P- N² -dG), and one from the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (C8-dG-PhIP), embedded in either full or ‘deletion’ duplexes (the partner nucleotide opposite the lesion is missing). All lesions adopt base-displaced intercalated conformations. Both full duplexes are thermodynamically destabilized and are excellent substrates of NER. However, the identical 10 R -(+)- cis-anti -B[ a ]P- N² -dG adduct in the deletion duplex dramatically enhances the thermal stability of this duplex, and is completely resistant to NER. Molecular dynamics simulations show that B[ a ]P lesion-induced distortion/destabilization is compensated by stabilizing aromatic ring system–base stacking interactions. In the C8-dG-PhIP-deletion duplex, the smaller size of the aromatic ring system and the mobile phenyl ring are less stabilizing and yield moderate NER efficiency. Thus, a partner nucleotide opposite the lesion is not an absolute requirement for the successful initiation of NER. Our observations are consistent with the hypothesis that carcinogen–base stacking interactions, which contribute to the local DNA stability, can prevent the successful insertion of an XPC β-hairpin into the duplex and the normal recruitment of other downstream NER factors.

The eukaryotic global genomic nucleotide excision repair (GG-NER) pathway is the major mechanism that removes most bulky and some nonbulky lesions from cellular DNA. There is growing evidence that certain DNA lesions are repaired slowly or are entirely resistant to repair in cells, tissues, and in cell extract model assay systems. It is well established that the eukaryotic DNA lesion-sensing proteins do not detect the damaged nucleotide, but recognize the distortions/destabilizations in the native DNA structure caused by the damaged nucleotides. In this article, the nature of the structural features of certain bulky DNA lesions that render them resistant to NER, or cause them to be repaired slowly, is compared to that of those that are good-to-excellent NER substrates. Understanding the structural features that distinguish NER-resistant DNA lesions from good NER substrates may be useful for interpreting the biological significance of biomarkers of exposure of human populations to genotoxic environmental chemicals. NER-resistant lesions can survive to replication and cause mutations that can initiate cancer and other diseases. Furthermore, NER diminishes the efficacy of certain chemotherapeutic drugs, and the design of more potent pharmaceuticals that resist repair can be advanced through a better understanding of the structural properties of DNA lesions that engender repair-resistance.