Abstract
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).
Highlights
To support and verify the relevance of the 50 -TCG sequence in the templates used in the experiments aimed at enzymatic translesion synthesis (TLS), we performed transcription mapping with the aid of SP6 and T7 RNA polymerases of the DNA–ACR adducts formed in both strands of the whole pSP73KB plasmid globally modified by ACR
0.01 for RNA synthesis by SP6 or T7 RNA polymerase, respectively, and by cisplatin at rb = 0.01 (Figure S1A)
RNA synthesis on the pSP73KB plasmid modified by monofunctional ACR and bifunctional cisplatin yielded fragments of defined sizes, which indicates that RNA synthesis on these templates was prematurely terminated
Summary
Platinum-based coordination compound cisplatin and its clinically approved and marketed analogs represent a class of successful chemotherapeutics globally used in medicine, especially as anticancer drugs [1]. They are characterized by a limited effect due to the limited types of tumors they are efficient against, intrinsic resistance, severe side effects, and, in particular, acquired resistance. Exposure of cells to genotoxic agents continually damages DNA. DNA damage tolerance is one of the most critical factors in developing resistance and potential mutagenicity of these platinum drugs [1,2,3].
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