Base Excision Repair of N6-Deoxyadenosine Adducts of 1,3-Butadiene

Abstract

The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N⁶-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N⁶-HB-dA), 1,N⁶-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N⁶-HMHP-dA), and N⁶,N⁶-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N⁶,N⁶-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2'-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (S)-N⁶-HB-dA, (R,S)-1,N⁶-HMHP-dA, and (R,R)-N⁶,N⁶-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (S)-N⁶-HB-dA > (R,R)-N⁶,N⁶-DHB-dA > (R,S)-1,N⁶-HMHP-dA. The extent of strand cleavage at the adduct site was decreased in the presence of BER inhibitor methoxyamine and by competitor duplexes containing known BER substrates. Similar strand cleavage assays conducted using several eukaryotic DNA glycosylases/lyases (AAG, Mutyh, hNEIL1, and hOGG1) have failed to observe correct incision products at the BD-dA lesion sites, suggesting that a different BER enzyme may be involved in the removal of BD-dA adducts in human cells.

Figure 1

Dynamics of DEB-induced 1,N⁶-HMHP-dA adducts in hamster V79 cells. Cells were treated with 100 μM DEB for 3 hours and allowed to repair the damage for 0.5–24 hours, followed by enzymatic digestion of genomic DNA and HPLC-ESI-MS/MS analysis of any lesions remaining.

Figure 2

Concentration dependent cleavage of DNA adducts by nuclear protein extracts from HT1080 cells. ³²P-endlabeled DNA 18-mer (5′-TCA TXG AAT CCT TCC CCC-3′) duplexes were incubated in 10 mM HEPES (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM EGTA and 0.1 mM DTT with increasing amounts of HT1080 nuclear extracts at 37 °C for 2 h. Samples were resolved on a 20% denaturing PAGE gel and visualized by phosphor imaging. (A) A representative PAGE gel for concentration dependent incision of 18-mer containing (R,S)-1,N⁶-HMHP-dA, and (B) volume analysis showing increasing amounts of incision products for BD-dA adducts with increasing amounts of nuclear extract.

Figure 3

Time-dependent repair of 1,3-butadiene-induced 2′-deoxyadenosine adducts by nuclear protein extracts from human fibrosarcoma (HT1080) cells. A representative PAGE gel of incision of 18-mer (5′-TCA TXG AAT CCT TCC CCC-3′) containing (R,S)-1,N⁶-HMHP-dA (A), time-dependent incision of 18-mers containing (S)-N⁶-HB-dA (B), (R,S)-1,N⁶-HMHP-dA (C), (R,R)-N⁶,N⁶-DHB-dA (D) in the presence (red lines) and absence (black lines) of BER inhibitor, methoxyamine. ³²P-endlabeled 18-mer DNA duplexes were incubated with 0.5 μg/μL nuclear extract in 10 mM HEPES (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM EGTA and 0.1 mM DTT at 37 °C. Aliquots of the reaction mixture were quenched at preselected time points, samples were resolved on a 20% denaturing PAGE gel and visualized by phosphor imaging. Volume analysis showed increasing amounts of incision products with increasing incubation time (n = 3).

Figure 4

Time course for excision of N⁶,N⁶-DHB-dA in the presence of nuclear protein extracts from human cells in comparison to known BER substrates (8-oxo-dG and 5F-dU). ³²P-endlabeled dsDNA 18-bp Duplex 2 containing site-specific adducts (50 nM, X = N⁶,N⁶-DHB-dA) was incubated with 0.5 μg/μL nuclear extract in 10 mM HEPES (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM EGTA and 0.1 mM DTT at 37 °C. Aliquots of the reaction mixture were quenched at preselected time points, and the samples were resolved on a 20% denaturing PAGE gel and visualized by phosphor-imaging. Volume analysis showed increasing amounts of incision products with increasing incubation time (n = 3).

Figure 5

MS/MS spectra of incision products detected following incubation of DNA duplex 2 containing site specific (R,S)-1,N⁶-HMHP-dA adducts with nuclear protein extracts from human fibrosarcoma (HT1080) cells.

Figure 6

Base excision repair assay using recombinant mammalian glycosylases: a representative PAGE gel of the repair assay in the presence of (A) Mutyh (B) hOGG1 (C) hAAG, and (D) hNEIL1 (edited and unedited) with 18-mer Duplexes 2 (AAG) and Duplex 3 (Mutyh, hOGG1 and hNEIL1) where X is paired opposite dT. Strand cleavage assays were performed under single-turnover conditions (STO, enzyme concentration > DNA substrate concentration).³²P-endlabeled double-stranded DNA substrates (10–50 nM) were incubated with 8–200-fold excess of each recombinant BER enzyme in a final volume of 20 μL. The assay buffer contained 20 mM Tris-HCl (pH 7.6), 10 mM EDTA, 100 μg/mL BSA, 30 mM NaCl, while the experiments using hNEIL1 (edited or unedited) included 60 mM NaCl. AAG reaction buffer was 20 mM Tris-HCl buffer (pH 7.8) containing 100 mM KCl, 5 mM β-mercaptoethanol, 2 mM EDTA, 1 mM EGTA, and 50 μg/mL BSA. Reactions were carried out at 37 °C for 60–180 min and analyzed as described in the methods section.

Scheme 1

Proposed mechanism for the formation of 1,3-butadiene (BD) induced 2′-deoxyadenosine adducts.

Scheme 2

DNA sequences and nucleobase lesions employed in the present study.

Scheme 3

Preparation of DNA strands containing site-specific BD-dA adducts by a post-oligomerization approach.