Conformational Insights into the Lesion and Sequence Effects for Arylamine-Induced Translesion DNA Synthesis: ¹⁹F NMR, Surface Plasmon Resonance, and Primer Kinetic Studies

Abstract

Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3′-next flanking base (G*CT vs G*CA; G*, FABP, N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl; FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair (JainV.HiltonB.LinB.PatnaikS.LiangF.DarianE.ZouY.MackerellA. D.Jr.ChoB. P.JainV.HiltonB.LinB.PatnaikS.LiangF.DarianE.ZouY.MackerellA. D.Jr.ChoB. P.23180767Nucleic Acids Res.201341869880). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo–) and Dpo4. We obtained dynamic 19F NMR spectra for two 19-mer modified templates during primer elongation: G*CA [d(5′-CTTACCATCG*CAACCATTC-3′)] and G*CT [d(5′-CTTACCATCG*CTACCATTC-3′)]. We found that lesion stacking is favored in the G*CT sequence compared to the G*CA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and G*CA > G*CT. Primer extension was stalled at (n) and near (n – 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct’s chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo– vs Dpo4). Steady-state kinetics analysis with Kf-exo– revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis.

Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

“…In the wedge (W-type) conformation ( 23 , 24 ), the C8-dG adduct also adopts the syn conformation, but steric clash at the lesion site, or Hoogsteen H-bonding with the opposing base, places the aryl moiety in the minor groove. The interplay between aryl ring size ( 18 ), adduct planarity ( 17 ) and DNA sequence ( 25 , 26 ) dictate conformational distribution, which are important factors that affect repair propensity ( 27 , 28 ) and mutational outcomes ( 29 – 33 ).…”

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

“…Different conformers of 2-(acetyl)aminofluorene-dG lesions have also been shown to play a role in their recognition and repair by NER [73][74] . While it is difficult to ascertain which conformation is prevalent in solution here for NPOM-DNA, base-displaced intercalated conformers can preferentially be stabilized in specific sequence contexts and be in equilibrium with major groove conformers [75][76][77] . Future studies are needed to reveal how Rad4binding changes the 3D structure of NPOM-DNA and how such structures impacts its repair by NER.…”

Real-Time Surface Plasmon Resonance Study of Biomolecular Interactions between Polymerase and Bulky Mutagenic DNA Lesions

“…FABP is similarly N -deacetylated, but it lacks a methylene bridge, resulting in a bulky twisted biphenyl moiety. 21 In other words, FABP may behave like FAAF at the replication fork of the template in the active site of a polymerase. In contrast to the unmodified control, modified adducts displayed a significant decrease (7–13-fold) in dissociation rate, with positive net stabilization energy ( Table S2 ).…”

“… 9 Other factors influencing adduct-induced mutations include topology, insertion of the nucleotide opposite the lesion site, and the characteristics of the polymerase. 21 , 22 …”

“…As a result, the choice of polymerases used in the present study was based largely on the experimental systems in our previous work. 7 , 8 , 21 Obviously, future SPR studies should be expanded to a range of Y-family bypass polymerases, which are more likely to be involved in replication of bulky DNA lesions.…”