Structural and thermodynamic insight into Escherichia coli UvrABC-mediated incision of cluster diacetylaminofluorene adducts on the NarI sequence

Abstract

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1*G2CG3*CC-3' (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.

Figure 1

Adduct structures and sequences. (a) Chemical structures of AAF and FAAF adducts; (b) fully paired 16-mer NarI duplexes; (c) major groove views of the central trimer segments of the B/S/W-conformer equilibrium of FAAF-modified mono-adduct duplexes. The modified dG and the complementary dC are shown in red and green sticks, respectively, and the aminofluorene moiety is highlighted with shiny grey CPK and the N-acetyl with pink CPK. In the B-type conformer, anti-glycosidic FAAF-dG maintains Watson-Crick hydrogen bonds, thereby placing the carcinogen moiety in the major groove. The carcinogens in the S- and W-conformers stack into the helix or wedged into the minor groove, respectively, with the modified dG in the syn conformation.

Figure 2

MALDI-TOF mass spectra of the hydrolysis fragments formed from exonuclease digestion of peak 4 (see Fig. S1) (a) the 3'- exonuclease digestion spectrum at 30 seconds (bottom), 5 (middle) and 15 (top) minutes. (b) the 5'-exonuclease digestion spectrum at 5 (bottom), 30 (middle) and 80 (top) minutes. Insets show the theoretical molecular weights of the fragments that should form after the 3’- and 5’-exonuclease digestion of peak 4 if oligodeoxynucleotide is modified at G₁ and G₃ positions.

Figure 3

CD and UV-melting profiles. (a) CD spectral overlays recorded at 20 °C and (b) UV-melting curves in 20 mM phosphate buffer containing 0.1 M NaCl at pH 7 of fully paired 16-mer NarI di-FAAF-adduct duplexes: G₁G₂ (green), G₁G₃ (blue), and G₂G₃ (red).

Figure 4

Comparative thermodynamic parameters histogram of NarI di-FAAF adduct duplexes; G₁G₂ (green), G₁G₃ (blue), and G₂G₃ (red). The ΔΔvalues represent; ΔΔH = ΔH (modified duplex) - ΔH (control duplex), TΔΔS= TΔS (modified duplex) - TΔS (control duplex), ΔΔG = ΔG (modified duplex) - ΔG (control duplex).

Figure 5

¹⁹F NMR spectra of di-FAAF-modified duplexes at different temperatures.

Figure 6

Representative conformations of the di-FAAF duplexes for the low energy glycosidic isomers obtained from the large clusters based on RMS Differences of the central 6 nucleotides (B: B-type; S-stacked). FAAF adducts are colored in red, DNA bases are colored in light blue except that G₁, G₂, G₃ and their complimentary cytosine bases are colored in green. Backbone and sugar rings are colored in grey. Number of frames for each cluster are in parentheses as listed in Supporting Information Table S2, which indicates the population of each cluster divided by the total number frames for each isomer (4000 in this study).

Figure 7

UvrABC nuclease incision kinetic results. (a) comparative relative incision rate histogram of FAAF modified mono- and di-adducts located at different positions calculated by considering NarI-G₂G₃ FAAF incision rate as 100%. (b) table showing the absolute incision rate of three di-adduct duplexes.