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

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2'-deoxyribo-5-hydroxyuridin (HdU) and 2'-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF-), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

• Klíčová slova
• 2’-deoxyribo-5-hydroxymethyl- uridin, 2’-deoxyribo-5-hydroxyuridin, DNA polymerases, microscale thermophoresis, oxidized nucleotides, translesion DNA synthesis,
• MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA biosyntéza   účinky léků   MeSH
• HIV-1 MeSH
• lidé MeSH
• mutageny chemie   metabolismus   farmakologie   MeSH
• oprava DNA MeSH
• oxidace-redukce MeSH
• oxidační stres * MeSH
• pentoxyl analogy a deriváty   chemie   metabolismus   farmakologie   MeSH
• poškození DNA MeSH
• pyrimidiny chemie   metabolismus   farmakologie   MeSH
• replikace DNA účinky léků   MeSH
• termodynamika MeSH
• uracil analogy a deriváty   chemie   metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 5-hydroxymethyluracil MeSH Prohlížeč
• 5-hydroxyuracil MeSH Prohlížeč
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• mutageny MeSH
• pentoxyl MeSH
• pyrimidiny MeSH
• Rad30 protein MeSH Prohlížeč
• uracil MeSH