Konstantin Kropachev

The human DNA repair factor XPC-HR23B distinguishes stereoisomeric benzo[a]pyrenyl-DNA lesions

Benzo[a]pyrene (B[a]P), a known environmental pollutant and tobacco smoke carcinogen, is metabolically activated to highly tumorigenic B[a]P diol epoxide derivatives that predominantly form N2‐guanine adducts in cellular DNA. Although nucleotide excision repair (NER) is an important cellular defense mechanism, the molecular basis of recognition of these bulky lesions is poorly understood. In order to investigate the effects of DNA adduct structure on NER, three stereoisomeric and conformationally different B[a]P‐N2‐dG lesions were site specifically incorporated into identical 135‐mer duplexes and their response to purified NER factors was investigated. Using a permanganate footprinting assay, the NER lesion recognition factor XPC/HR23B exhibits, in each case, remarkably different patterns of helix opening that is also markedly distinct in the case of an intra‐strand crosslinked cisplatin adduct. The different extents of helix distortions, as well as differences in the overall binding of XPC/HR23B to double‐stranded DNA containing either of the three stereoisomeric B[a]P‐N2‐dG lesions, are correlated with dual incisions catalyzed by a reconstituted incision system of six purified NER factors, and by the full NER apparatus in cell‐free nuclear extracts.

Resistance of bulky DNA lesions to nucleotide excision repair can result from extensive aromatic lesion–base stacking interactions

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 (10R-(+)-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 10R-(+)-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.

Structural basis for the recognition of diastereomeric 5′,8-cyclo-2′-deoxypurine lesions by the human nucleotide excision repair system

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.

Nucleotide excision repair of 2-acetylaminofluorene- and 2-aminofluorene-(C8)-guanine adducts: molecular dynamics simulations elucidate how lesion structure and base sequence context impact repair efficiencies

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 G1, G2 or G3 in the duplex sequence (5′-CTCG1G2CG3CCATC-3′) containing the NarI mutational hot spot. Furthermore, the dG-C8-AAF adduct is a better substrate of NER than dG-C8-AF in all three NarI 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.

Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similar relative NER incision efficiencies for ∼65% of these substrates; the other cases deviate mostly by ∼30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER.

Adenine-DNA adducts derived from the highly tumorigenic Dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not.

The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N(2)-dG) and adenine (N(6)-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N(2)-dG linkage site is ∼35 times more susceptible to NER dual incisions than the stereochemically identical N(6)-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar but somewhat smaller effect (factor of ∼15) is observed. The striking resistance of the bulky N(6)-dA in contrast to the modest to good susceptibilities of the N(2)-dG adducts to NER is interpreted in terms of the balance between lesion-induced DNA distorting and DNA stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA.

Base Flipping Free Energy Profiles for Damaged and Undamaged DNA

Lesion-induced thermodynamic destabilization is believed to facilitate β-hairpin intrusion by the human XPC/hHR23B nucleotide excision repair (NER) recognition factor, accompanied by partner-base flipping, as suggested by the crystal structure of the yeast orthologue (Min, J. H., and Pavletich, N. P. (2007) Nature 449, 570-575). To investigate this proposed mechanism, we employed the umbrella sampling method to compute partner base flipping free energies for the repair susceptible 14R (+)-trans-anti-DB[a,l]P-N(2)-dG modified duplex 11-mer, derived from the fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene, and for the undamaged duplex. Our flipping free energy profiles show that the adduct has a lower flipping barrier by ∼7.7 kcal/mol, consistent with its thermally destabilizing impact on the damaged DNA duplex and its susceptibility to NER.

Generation of guanine-thymine cross-links in human cells by one-electron oxidation mechanisms.

The one-electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation produces cross-links between guanine and thymine bases (G*-T*), characterized by a covalent bond between C8 guanine (G*) and N3 thymine (T*) atoms. The DNA lesions were quantified by isotope dilution LC-MS/MS methods in the multiple reaction-monitoring mode using isotopically labeled [(15)N, (13)C]-nucleotides as internal standards. Among several known pyrimidine and 8-oxo-7,8-dihydroguanine lesions, the G*-T* cross-linked lesions were detected at levels of ~0.21 and 1.19 d(G*-T*) lesions per 10(6) DNA bases at laser intensities of 50 and 280 mJ/cm(2)/pulse, respectively.

Structural, energetic and dynamic properties of guanine(C8)-thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair.

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742–755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5′-d(…G*pT*…) and 5′-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson–Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.

Exploring Damage Recognition Models in Prokaryotic Nucleotide Excision Repair with a Benzo(a)pyrene-Derived Lesion in UvrB

The UvrB protein is a central unit for damage recognition in the prokaryotic nucleotide excision repair system, which excises bulky DNA lesions. We have utilized molecular modeling and MD simulations based on crystal structures, mutagenesis, and fluorescence data, to model the 10R-(+)-cis-anti-B[a]P-N2-dG lesion, derived from the tumorigenic (+)-anti-B[a]PDE metabolite of benzo[a]pyrene, at different locations on the inner and outer strand in UvrB. Our results suggest that this lesion is accommodated on the inner strand where it might translocate through the tunnel created by the beta-hairpin and UvrB domain 1B and ultimately could be housed in the pocket behind the beta-hairpin prior to excision by UvrC. Lesions that vary in size and shape may be stopped at the gate to the tunnel, within the tunnel, or in the pocket when UvrC initiates excision. Common features of beta-hairpin intrusion between the two DNA strands and nucleotide flipping manifested in structures of prokaryotic and eukaryotic NER lesion recognition proteins are consistent with common recognition mechanisms, based on lesion-induced local thermodynamic distortion/destabilization and nucleotide flipping.