Probing the Effect of Bulky Lesion-Induced Replication Fork Conformational Heterogeneity Using 4-Aminobiphenyl-Modified DNA

Abstract

Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations have been implicated in different mutagenic and repair outcomes. However, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce, presumably due to the structural dynamics of DNA lesions at the replication fork. It is difficult to capture the exact nature of replication inhibition by existing end-point assays, which usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. We previously reported very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG₁*G₂T [67%B:33%S] and TG₁G₂*T [100%B], G*, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl) (Cai et al. Nucleic Acids Research, 46, 6356-6370 (2018)). In the present study, we attempted to correlate the in vitro inhibition of polymerase activity to different conformations from a single FABP-modified DNA lesion. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B).

Keywords: 4-aminobiphenyl; Klenow fragment; bulky DNA lesion; conformational heterogeneity; steady state enzyme kinetics; surface plasmon resonance (SPR) binding kinetics.

Figure 1

(a) Two major conformational views of dG-C8-FABP [N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl]: B-, and S-conformers. Fluorine-labeled ABP (FABP)-red, modified dG-blue, complementary C-orange. (b) The chemical structure of dG-C8-FABP.

Figure 2

(a) Hairpin template−primer oligonucleotide constructs of 85-mer G₁ and 84-mer G₂ for Kf-exo⁻, H denotes 3′-dideoxy-nucleotide; (b) Sensorgrams of binary complexes of Kf-exo⁻ with 85-mer and 84-mer unmodified control, G₁-FABP and G₂-FABP modified sequences (1:1 binding fitted curves are overlaid as black lines); (c) K_a (K_a-modified/K_a-contol) and K_d (K_d-modified/K_d-contol) ratio, G₁ and G₂ represent the ratio of 85-mer FABP-G₁/85-mer-control and 84-mer FABP-G₂/84-mer-control, respectively.

Figure 3

DNA replication models for (a) FABP-modified G₁* (TG*GT) templates and (b) FABP-modified G₂* (TGG*T) templates. n: lesion site. Lineweaver–Burk model of DNA synthesis catalyzed by Kf-exo⁻ at (c) 10-mer, G₁-FABP and (d) 9-mer G₂-FABP lesion sites with dCTP at steady-state. The G₁-FABP against with Kf–exo^– adopts a competitive inhibitor model, whereas the behavior of TG₁G₂*T shows as non-competitive (see text).

Figure 4

Sensorgrams of the ternary Kf-exo⁻ complexed with (a) 85-mer control, (b) 85-mer TG₁[FABP]G₂T, (c) 84-mer control and (d) 84-mer TG₁G₂[FABP]T sequences in the presence of dNTPs (1:1 binding fitted curves are overlaid as black lines).