Marina Kolbanovskiy

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.

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.

Base and Nucleotide Excision Repair of Oxidatively Generated Guanine Lesions in DNA.

The well known biomarker of oxidative stress, 8-oxo-7,8-dihydroguanine, is more susceptible to further oxidation than the parent guanine base and can be oxidatively transformed to the genotoxic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. Incubation of 135-mer duplexes with single Sp or Gh lesions in human cell extracts yields a characteristic nucleotide excision repair (NER)-induced ladder of short dual incision oligonucleotide fragments in addition to base excision repair (BER) incision products. The ladders were not observed when NER was inhibited either by mouse monoclonal antibody (5F12) to human XPA or in XPC−/− fibroblast cell extracts. However, normal NER activity appeared when the XPC−/− cell extracts were complemented with XPC-RAD23B proteins. The Sp and Gh lesions are excellent substrates of both BER and NER. In contrast, 5-guanidino-4-nitroimidazole, a product of the oxidation of guanine in DNA by peroxynitrite, is an excellent substrate of BER only. In the case of mouse embryonic fibroblasts, BER of the Sp lesion is strongly reduced in NEIL1−/− relative to NEIL1+/+ extracts. In summary, in human cell extracts, BER and NER activities co-exist and excise Gh and Sp DNA lesions, suggesting that the relative NER/BER product ratios may depend on competitive BER and NER protein binding to these lesions.

Nuclear magnetic resonance studies of an N2-guanine adduct derived from the tumorigen dibenzo[a,l]pyrene in DNA: impact of adduct stereochemistry, size, and local DNA sequence on solution conformations.

The dimensions and arrangements of aromatic rings (topology) in adducts derived from the reactions of polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites with DNA influence the distortions and stabilities of double-stranded DNA, and hence their recognition and processing by the human nucleotide excision repair (NER) system. Dibenzo[a,l]pyrene (DB[a,l]P) is a highly tumorigenic six-ring PAH, which contains a nonplanar and aromatic fjord region that is absent in the structurally related bay region five-ring PAH benzo[a]pyrene (B[a]P). The PAH diol epoxide–DNA adducts formed include the stereoisomeric 14S and 14Rtrans-anti-DB[a,l]P-N2-dG and the stereochemically analogous 10S- and 10R-B[a]P-N2-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N2-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N2-dG adduct in two different sequence contexts using NMR methods with distance-restrained molecular dynamics simulations. In duplexes with dC opposite the adduct deleted, a well-resolved base-displaced intercalative adduct conformation can be observed. In full duplexes, in contrast to the intercalated 14R stereoisomeric adduct, the bulky DB[a,l]P residue in the 14S adduct is positioned in a greatly widened and distorted minor groove, with significant disruptions and distortions of base pairing at the lesion site and two 5′-side adjacent base pairs. These unique structural features are significantly different from those of the stereochemically analogous but smaller B[a]P-dG adduct. The greater size and different topology of the DB[a,l]P aromatic ring system lead to greater structurally destabilizing DNA distortions that are partially compensated by stabilizing DB[a,l]P-DNA van der Waals interactions, whose combined effects impact the NER response to the adduct. These structural results broaden our understanding of the structure–function relationship in NER.

Inefficient nucleotide excision repair in human cell extracts of the N-(deoxyguanosin-8-yl)-6-aminochrysene and 5-(deoxyguanosin-N(2)-yl)-6-aminochrysene adducts derived from 6-nitrochrysene.

Ubiquitous environmental agents [e.g., polynuclear aromatic hydrocarbons (PAHs) and their nitrated derivatives (NO(2)-PAHs)] that are known to induce mammary cancer in rodents are regarded as potential human risk factors for inducing analogous human cancers. Although 6-nitrochrysene (6-NC) is less abundant than other NO(2)-PAHs in the environment, it is the most potent mammary carcinogen in the rat; its carcinogenic potency is not only higher than that of the carcinogenic PAH, benzo[a]pyrene (B[a]P), but also of the well-known carcinogenic heterocylic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). Studies in rats and in vitro assays have indicated that 6-NC can be activated by simple nitroreduction leading to the formation of 6-hydroxylaminochrysene (N-OH-6-AC); this metabolite yielded N-(deoxyguanosin-8-yl)-6-aminochrysene (N-[dG-8-yl]-6-AC) and 5-(deoxyguanosin-N(2)-yl)-6-aminochrysene (5-[dG-N(2)-yl]-6-AC. These lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. However, nothing is known about the susceptibility of these adducts to nucleotide excision repair (NER), the major cellular repair system that removes bulky adducts. In order to address this issue, we synthesized the N-(dG-8-yl)-6-AC and 5-(dG- N(2)-yl)-6-AC lesions and site-specifically inserted these lesions into 135-mer DNA duplexes. These constructs were incubated with NER-competent nuclear extracts from human HeLa cells. The efficiency of repair of these lesions was ∼ 8 times less efficient than that in the case of the well-known and excellent substrate of NER, the intrastrand cross-linked cis-diaminodichloroplatinum II adduct in double-stranded DNA (cis-Pt), but similar to N(2)-dG adducts derived from the (+)-bay region diol epoxide of B[a]P [(+)-trans-B[a]P-N(2)-dG]. The results support the hypothesis that the N-(dG-8-yl)-6-AC and 5-(dG-N(2)-yl)-6-AC lesions may be slowly repaired and thus persistent in mammalian tissue which could, in part, account for the potent tumorigenic activity of 6-NC in the rat mammary gland.