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).

• Keywords
• DNA polymerases, antitumor, cytotoxic, drug resistance, lesion bypass, metal–intercalator, microscale thermophoresis, platinum–acridine, thermodynamic, translesion synthesis,
• MeSH
• DNA Adducts chemistry   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• Intercalating Agents chemistry   MeSH
• Humans MeSH
• Urea analogs & derivatives   chemistry   MeSH
• DNA Repair * MeSH
• Organoplatinum Compounds chemistry   MeSH
• DNA Damage * MeSH
• DNA Replication MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1-(2-(acridin-9-ylamino)ethyl)-1,3-dimethylthiourea MeSH Browser
• DNA Adducts MeSH
• DNA-Directed DNA Polymerase MeSH
• Intercalating Agents MeSH
• Urea MeSH
• Organoplatinum Compounds 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.

• Keywords
• DNA polymerases, antitumor, microscale thermophoresis, platinum-acridine, translesion DNA synthesis,
• MeSH
• DNA Adducts chemistry   genetics   metabolism   MeSH
• Acridines chemistry   pharmacology   MeSH
• Biocatalysis MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA biosynthesis   MeSH
• Guanine metabolism   MeSH
• Catalysis MeSH
• Nucleotides genetics   metabolism   MeSH
• DNA Repair MeSH
• DNA Replication MeSH
• Platinum Compounds chemistry   pharmacology   MeSH
• Thermal Diffusion MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Adducts MeSH
• Acridines MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Guanine MeSH
• Nucleotides MeSH
• Platinum Compounds MeSH

We studied the effect of antitumor cisplatin and inefficient transplatin on the structure and stability of G quadruplexes formed by the model human telomere sequence 5'-GGG(TTAGGG)(3)-3' using circular dichroism, UV-monitored thermal denaturation, and gel electrophoresis. In addition, to investigate whether there is a connection between the ability of cisplatin or transplatin to affect telomerase activity and stability of G quadruplexes, we also used a modified telomere repeat amplification protocol assay that uses an oligonucleotide substrate for telomerase elongation susceptible to forming a G quadruplex. The results indicate that cisplatin is more efficient than transplatin in disturbing the quadruplex structure, thereby precluding telomeric sequences from forming quadruplexes. On the other hand, the results of this work also demonstrate that in absence of free platinum complex, DNA adducts of antitumor cisplatin inhibit telomerase catalysis, so the mechanism underlying this inhibition does not involve formation of the G quadruplexes which are not elongated by telomerase.

• MeSH
• DNA Adducts drug effects   genetics   metabolism   MeSH
• Biocatalysis MeSH
• Circular Dichroism MeSH
• Cisplatin chemistry   pharmacology   MeSH
• Nucleic Acid Denaturation MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• G-Quadruplexes drug effects   MeSH
• Humans MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Base Sequence MeSH
• Spectrophotometry, Ultraviolet MeSH
• Nucleic Acid Amplification Techniques MeSH
• Telomerase antagonists & inhibitors   metabolism   MeSH
• Telomere chemistry   genetics   MeSH
• Transition Temperature MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Cisplatin MeSH
• Antineoplastic Agents MeSH
• Telomerase MeSH
• transplatin MeSH Browser

Clinically ineffective transplatin [trans-diamminedichloridoplatinum(II)] is used in the studies of the structure-pharmacological activity relationship of platinum compounds. In addition, a number of transplatin analogs exhibit promising toxic effects in several tumor cell lines including those resistant to conventional antitumor cisplatin. Moreover, transplatin-modified oligonucleotides have been shown to be effective modulators of gene expression. Owing to these facts and because DNA is also considered the major pharmacological target of platinum complexes, interactions between transplatin and DNA are of great interest. We examined, using biophysical and biochemical methods, the stability of 1,3-GNG intrastrand cross-links (CLs) formed by transplatin in short synthetic oligodeoxyribonucleotide duplexes and natural double-helical DNA. We have found that transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand CLs are relatively stable. We show that the stability of 1,3-GNG intrastrand CLs of transplatin correlates with the extent of conformational distortion and thermodynamic destabilization induced in double-helical DNA by this adduct.

• MeSH
• Biophysical Phenomena * MeSH
• Cisplatin metabolism   MeSH
• DNA chemistry   genetics   metabolism   MeSH
• Calorimetry MeSH
• Nucleic Acid Conformation MeSH
• Oligodeoxyribonucleotides chemistry   genetics   metabolism   MeSH
• Cross-Linking Reagents metabolism   MeSH
• Base Sequence MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cisplatin MeSH
• DNA MeSH
• Oligodeoxyribonucleotides MeSH
• Cross-Linking Reagents MeSH
• transplatin MeSH Browser