Thermodynamic Insights by Microscale Thermophoresis into Translesion DNA Synthesis Catalyzed by DNA Polymerases Across a Lesion of Antitumor Platinum–Acridine Complex

Abstract

Translesion synthesis (TLS) through DNA adducts of antitumor platinum complexes has been an interesting aspect of DNA synthesis in cells treated with these metal-based drugs because of its correlation to drug sensitivity. We utilized model systems employing a DNA lesion derived from a site-specific monofunctional adduct formed by antitumor [PtCl(en)(L)](NO3)2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine) at a unique G residue. The catalytic efficiency of TLS DNA polymerases, which differ in their processivity and fidelity for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the adduct of AMD, was investigated. For a deeper understanding of the factors that control the bypass of the site-specific adducts of AMD catalyzed by DNA polymerases, we also used microscale thermophoresis (MST) to measure the thermodynamic changes associated with TLS across a single, site-specific adduct formed in DNA by AMD. The relative catalytic efficiency of the investigated DNA polymerases for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the AMD adduct, was reduced. Nevertheless, incorporation of the correct C opposite the G modified by AMD of the template strand was promoted by an increasing thermodynamic stability of the resulting duplex. The reduced relative efficiency of the investigated DNA polymerases may be a consequence of the DNA intercalation of the acridine moiety of AMD and the size of the adduct. The products of the bypass of this monofunctional lesion produced by AMD and DNA polymerases also resulted from the misincorporation of dNTPs opposite the platinated G residues. The MST analysis suggested that thermodynamic factors may contribute to the forces that governed enhanced incorporation of the incorrect dNTPs by DNA polymerases.

Processing and Bypass of a Site-Specific DNA Adduct of the Cytotoxic Platinum–Acridinylthiourea Conjugate by Polymerases Involved in DNA Repair: Biochemical and Thermodynamic Aspects

“…Our results complement the picture of the action of platinum conjugates with an intercalating acridine ligand at the level of DNA damage and subsequent biological consequences; improved cytotoxic response of tumour cells to treatment with an “enhanced” AMD conjugate, where thiourea was replaced by an amidine group and where TLS behind the AMD adduct was strongly suppressed [ 43 , 51 , 57 ]. Thus, it would be possible to explain the differences between the cytotoxicity of the ACR conjugate and its AMD analogue by their different ability to overcome tumour cell resistance caused by lesion tolerance and bypass.…”

“…Because MST has proven to be a good method for determining the TD parameters of DNA lesions [ 50 , 51 ], the appropriate template, unmodified or modified by ACR, was paired with a set of primers (from n − 1 to n + 2) (shown in Figure 1 B and Figure 5 A) to simulate translesion synthesis [ 28 , 48 , 50 , 51 ]. The primers were prolonged on the 5′ site by four nucleotides to eliminate the possible effects of Cy5 fluorophore on hybridized DNA duplexes.…”

“…The K d values of the primer/template hybridization (for the given appropriate temperature in the temperature range of 22–45 °C) were determined and then plotted in a Van ’t Hoff plot as ln( K a ) vs. 1/ T ( Figure S3 ). This temperature dependence of the association constant K a (which equals 1/ K d ) was used to calculate the thermodynamic parameters Δ H , Δ S , and Δ G (see Figure 5 , Figure S3 and Table S3 ) as described in the experimental part and the previously published work [ 49 , 50 , 51 ].…”

“…Microscale thermophoresis (MST) is a suitable technique for obtaining thermodynamic (TD) parameters of modified oligonucleotides [ 49 , 50 , 51 ]. We measured the DNA hybridization equilibrium binding constant K a of a 15-mer oligonucleotide and a Cy5-labelled complementary primer over a range of temperatures (22–45 °C) using a Monolith NT115 Pico instrument.…”

“…We investigated in this study the DNA adduct of ACR in terms of its effect on thermodynamic (TD) parameters describing the stability of DNA duplexes in the place of its origin or its immediate vicinity. We used in these experiments microscale thermophoresis (MST) which has proven to be a useful technique for obtaining TD parameters of damaged DNA [ 49 , 50 , 51 ]. The results of these thermodynamic experiments simulating TLS were compared with those of enzymatic TLS across a site-specific DNA adduct of ACR (an ability of the ACR adduct to block DNA synthesis by various DNA polymerases and/or cause a mutation) in a cell-free medium.…”