AlkB homologue 2-mediated repair of ethenoadenine lesions in mammalian DNA

Abstract

Endogenous formation of the mutagenic DNA adduct 1,N(6)-ethenoadenine (epsilon A) originates from lipid peroxidation. Elevated levels of epsilon A in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for epsilon A lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair epsilon A lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2(-/-) mice indicates that mABH2 is the principal dioxygenase for epsilon A repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of epsilon A lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of epsilon A, comprise the cellular defense against epsilon A lesions.

Figure 1. DNA substrates for εA and 3meC repair and εA repair activities of AlkB and hABH2

(A) To create εA- and 3meC-containing substrates, 49 mer oligonucleotides were modified with an εA in position 24, or 3meC in position 26, radioactively labeled at the 5’-end and hybridized to a complementary strand. The modified bases are located in a DpnII restriction site such that this enzyme cleaves the substrate only if the etheno/methyl group is removed. Unrepaired substrate appears as a band of 49 nt, whereas repaired and cleaved substrate will appear as a 22 nt band following denaturing PAGE. (B) Activities of purified AlkB, hABH2 and ANPG on εA in ss DNA and ds DNA. DNA substrates were incubated with purified enzymes as indicated, digested with DpnII, or treated with 0.1M NaOH/30 min 90°C if reacted with ANPG, and separated by 20% denaturing PAGE. Labeled DNA was visualized by phosphorimaging. Untreated DNA substrates were incubated with DpnII as negative control. (C) Activities of purified hABH3 on εA in ss DNA and ds DNA. Same reaction conditions as in (B). (D) Reactions as in B. Prior to incubation, cold DNA substrates were added

Figure 2. Single-turnover kinetics for repair of εA

Substrate DNA (2 nM) was incubated with three different (twice to 100 times higher) concentrations of hABH2 and AlkB to measure product formation as a function of time. Each value represents the average of three independent measurements. (A) Plots of product formation as a function of time using 1000 nM enzyme, together with a curve fit (r2 values: hABH2, 0.99651; AlkB, 0.97915) to the experimental data [see (27)], to determine the active substrate concentration. (B and C) Linear regression curves (r2 values hABH2: 20 nM, 0.94361; 200 nM, 0.99617; 1000 nM, 0.96471; r2 values AlkB: 20 nM, 0.94061; 200 nM, 0.97327; 1000 nM, 0.92739) showing the initial product formation as a function of time (where the reaction rate is determined by their slopes) at the different enzyme concentrations. (D) Calculated k values (determined from the value of the slope of the curves presented in B divided by the active enzyme concentration) as a function of total enzyme concentration together with a curve fit (r2 values: hABH2, 0.99533; AlkB, 0.99631) to the experimental data (27).

Figure 3. Repair of εA and 3meC in wild-type cell extracts

Repair of εA (panel A) and 3meC (panel B, positive control) in fractionated wild-type extract is demonstrated with DpnII cleavage, as described in Figure 1. Repair of εA and 3meC was measured in freshly prepared protein fractions corresponding to 0.75 μg total protein. Repair activities peaked in fractions 29–31 and were completely dependent on mABH2 as no activity was observed in fractions from mABH2^−/− mice (C and D). Right panels A and B indicate the proportion of the substrate (from the gels shown) that has been repaired and cleaved by DpnII. Details and substrate as in Figure 1. NE: nuclear extract; F: flow through.

Figure 4. Survival of CAA- or MMS-treated M13 ssDNA phage in alkB mutant bacteria producing different AlkB proteins

HK82/F′ alkB bacteria carrying expression plasmids for the proteins indicated were infected with ssDNA phage M13 treated with the indicated concentrations of CAA (A) or MMS (B). Formation of progeny phage was assessed by counting plaques, and values are expressed relative to untreated M13. Error bars represent the standard deviation of triplicate samples (The apparent non-symmetry of the error bars is due to the logarithmic scale).

Figure 5. Effect of CAA on growth and viability of wild-type, mABH2^−/− and ANPG^−/− MEF cells

Wild-type cells, mABH2^−/− and ANPG^−/− cells were used at passages 16 to 34. Cells were seeded in 96-well plates with approximately 3000 cells/well. After 22 hours the cells were treated with 0-20 μM CAA for 2 hours, washed twice with PBS, before fresh media was added. Cell survival was measured after 48 h. Results are expressed as cell numbers in CAA-treated cultures compared to untreated controls. Cell numbers were measured using the MTT assay (Roche). Untreated wild-type, mABH2^−/− (A) and ANPG^−/− (B) cell lines had a density of about 60% after 48 hours. Values presented are average of five to ten independent experiments, and the standard deviations are always less than 10%.

Figure 6. Age-dependent accumulation of εA in wild-type, ABH2^−/−, ABH3^−/− and ANPG^−/− mice

Histogram graph showing the quantification of εA DNA lesions in liver tissue from wild type, mABH2^−/− and mABH3^−/− mice ranging in age from 4 to 12-months (panel A) and wild-type and ANPG^−/− mice ranging in age from 2 to 24 months (panel B). Lesions were quantified using a highly sensitive HPLC-tandem mass spectrometric (MS-MS) method (30). A minimum of 2 and a maximum of 5 mice were used per time point/per genotype. * p<0.05 24 months ANPG^−/− mice compared with 24 months old wild-type or 2 months old ANPG^−/− mice.