DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work performed in cell-free media, we studied the ability of selected DNA polymerases to overcome a monofunctional adduct of the cytotoxic/antitumor platinum-acridinylthiourea conjugate [PtCl(en)(L)](NO3)2 (en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) (ACR) in its favored 5'-CG sequence. We focused on how a single site-specific ACR adduct with intercalation potency affects the processivity and fidelity of DNA-dependent DNA polymerases involved in translesion synthesis (TLS) and repair. The ability of the G(N7) hybrid ACR adduct formed in the 5'-TCGT sequence of a 24-mer DNA template to inhibit the synthesis of a complementary DNA strand by the exonuclease-deficient Klenow fragment of DNA polymerase I (KFexo-) and human polymerases eta, kappa, and iota was supplemented by thermodynamic analysis of the polymerization process. Thermodynamic parameters of a simulated translesion synthesis across the ACR adduct were obtained by using microscale thermophoresis (MST). Our results show a strong inhibitory effect of an ACR adduct on enzymatic TLS: there was only small synthesis of a full-length product (less than 10%) except polymerase eta (~20%). Polymerase eta was able to most efficiently bypass the ACR hybrid adduct. Incorporation of a correct dCMP opposite the modified G residue is preferred by all the four polymerases tested. On the other hand, the frequency of misinsertions increased. The relative efficiency of misinsertions is higher than that of matched cytidine monophosphate but still lower than for the nonmodified control duplex. Thermodynamic inspection of the simulated TLS revealed a significant stabilization of successively extended primer/template duplexes containing an ACR adduct. Moreover, no significant decrease of dissociation enthalpy change behind the position of the modification can contribute to the enzymatic TLS observed with the DNA-dependent, repair-involved polymerases. This TLS could lead to a higher tolerance of cancer cells to the ACR conjugate compared to its enhanced analog, where thiourea is replaced by an amidine group: [PtCl(en)(L)](NO3)2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine).

• Klíčová slova
• DNA polymerases, antitumor, cytotoxic, drug resistance, lesion bypass, metal–intercalator, microscale thermophoresis, platinum–acridine, thermodynamic, translesion synthesis,
• MeSH
• adukty DNA chemie   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• interkalátory chemie   MeSH
• lidé MeSH
• močovina analogy a deriváty   chemie   MeSH
• oprava DNA * MeSH
• organoplatinové sloučeniny chemie   MeSH
• poškození DNA * MeSH
• replikace DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 1-(2-(acridin-9-ylamino)ethyl)-1,3-dimethylthiourea MeSH Prohlížeč
• adukty DNA MeSH
• DNA-dependentní DNA-polymerasy MeSH
• interkalátory MeSH
• močovina MeSH
• organoplatinové sloučeniny MeSH

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.

• Klíčová slova
• DNA polymerases, antitumor, microscale thermophoresis, platinum-acridine, translesion DNA synthesis,
• MeSH
• adukty DNA chemie   genetika   metabolismus   MeSH
• akridiny chemie   farmakologie   MeSH
• biokatalýza MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA biosyntéza   MeSH
• guanin metabolismus   MeSH
• katalýza MeSH
• nukleotidy genetika   metabolismus   MeSH
• oprava DNA MeSH
• replikace DNA MeSH
• sloučeniny platiny chemie   farmakologie   MeSH
• tepelná difuze MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adukty DNA MeSH
• akridiny MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• guanin MeSH
• nukleotidy MeSH
• sloučeniny platiny MeSH