Human and E.Coli excinucleases are affected differently by the sequence context of acetylaminofluorene-guanine adduct

Department of Biochemistry and Biophysics, University of North Carolina, School of Medicine, Chapel Hill.
Nucleic Acids Research (Impact Factor: 9.11). 12/1994; 22(23):4869-71. DOI: 10.1093/nar/22.23.4869
Source: PubMed

ABSTRACT Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE). The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report [Seeberg, E., and Fuchs, R.P.P. (1990) Proc. Natl Acad. Sci. USA 87, 191-194]. The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68. These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.

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    • "The relative NER rates for the dG-C8-AAF G1* and dG-C8-AAF G3* duplexes are the same within experimental error as well and are about 1.8 times smaller than the dG-C8-AAF G2* rates. It is noteworthy that our results for the dG-C8-AAF adduct with HeLa cell extracts follow the same trend as those obtained earlier by Mu et al. (25), with the dG-C8-AAF G2* duplex being repaired significantly more efficiently than the dG-C8-AAF G1* and dG-C8-AAF G3* duplexes. Thermal melting studies (Supplementary Figure S2) also show that the dG-C8-AAF adducts destabilize double-stranded DNA more extensively than the dG-C8-AF adducts as has been observed previously (33,68). "
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    ABSTRACT: 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 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.
    Nucleic Acids Research 08/2012; 40(19):9675-90. DOI:10.1093/nar/gks788 · 9.11 Impact Factor
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    • "Certain bulky DNA adducts, including N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) (45), N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP) (46), 10S-(−) (47) and 10R-(+)-cis-anti-B[a]P-N2-dG (34), which adopt base-displaced intercalated conformations in full duplexes have been shown to be well repaired in human HeLa cell extract assays (13,15,48,49) or in the prokaryotic UvrABC system (50). Although each adduct has its unique structural features, common structural properties of base-displaced intercalation appear to offer similar recognition elements to the NER system. "
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    ABSTRACT: 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-N(2)-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-N(2)-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.
    Nucleic Acids Research 07/2011; 39(20):8752-64. DOI:10.1093/nar/gkr537 · 9.11 Impact Factor
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    • "Although this approach is limited in terms of accessible sequences and yields to heterogeneous mixtures of adducts (11,12,17), it has been used in conjunction with extensive high-performance liquid chromatography (HPLC) purification to prepare defined single dG-AAF adducts in oligonucleotides containing up to three guanine residues (18–21). Biochemical studies using these well-defined damaged oligonucleotides have allowed invaluable insights into the impact of these lesions on DNA structure (22–24), replication (24–26), mutagenesis (14,15,20,27–29) and repair (6,19,30,31). Despite these advances, the generation of AAF-containing oligonucleotides by solid-phase DNA synthesis would greatly facilitate further biological investigations. "
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    ABSTRACT: Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified 'ultra-mild' DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.
    Nucleic Acids Research 02/2005; 33(6):1961-9. DOI:10.1093/nar/gki335 · 9.11 Impact Factor
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