The Stability of a Model Substrate for Topoisomerase 1-Mediated DNA Religation Depends on the Presence of Mismatched Base Pairs

Topoisomerase 1 (Top1) enzymes regulate DNA superhelicity by forming covalent cleavage complexes that undergo controlled rotation. Substitution of nucleoside analogs at the +1 position of the DNA duplex relative to the Top1 cleavage site inhibits DNA religation. The reduced efficiency for Top1-mediated religation contributes to the anticancer activity of widely used anticancer drugs including fluoropyrimidines and gemcitabine. In the present study, we report that mismatched base pairs at the +1 position destabilize the duplex DNA components for a model Top1 cleavage complex formation even though one duplex component does not directly include a mismatched base pair. Molecular dynamics simulations reveal G-dU and G-FdU mismatched base pairs, but not a G-T mismatched base pair, increase flexibility at the Top1 cleavage site, and affect coupling between the regions required for the religation reaction to occur. These results demonstrate that substitution of dT analogs into the +1 position of the non-scissile strand alters the stability and flexibility of DNA contributing to the reduced efficiency for Top1-mediated DNA religation. These effects are inherent in the DNA duplex and do not require formation of the Top1:DNA complex. These results provide a biophysical rationale for the inhibition of Top1-mediated DNA religation by nucleotide analog substitution

Stabilization of a G-Quadruplex from Unfolding by Replication Protein A Using Potassium and the Porphyrin TMPyP4

Replication protein A (RPA) plays an essential role in DNA replication by binding and unfolding non-canonical single-stranded DNA (ssDNA) structures. Of the six RPA ssDNA binding domains (labeled A-F), RPA-CDE selectively binds a G-quadruplex forming sequence (5′-TAGGGGAAGGGTTGGAGTGGGTT-3′ called Gq23). In K+, Gq23 forms a mixed parallel/antiparallel conformation, and in Na+ Gq23 has a less stable (TM lowered by ∼20°C), antiparallel conformation. Gq23 is intramolecular and 1D NMR confirms a stable G-quadruplex structure in K+. Full-length RPA and RPA-CDE-core can bind and unfold the Na+ form of Gq23 very efficiently, but complete unfolding is not observed with the K+ form. Studies with G-quadruplex ligands, indicate that TMPyP4 has a thermal stabilization effect on Gq23 in K+, and inhibits complete unfolding by RPA and RPA-CDE-core. Overall these data indicate that G-quadruplexes present a unique problem for RPA to unfold and ligands, such as TMPyP4, could possibly hinder DNA replication by blocking unfolding by RPA

Identification of the DNA-Binding Domains of Human Replication Protein A That Recognize G-Quadruplex DNA

Replication protein A (RPA), a key player in DNA metabolism, has 6 single-stranded DNA-(ssDNA-) binding domains (DBDs) A-F. SELEX experiments with the DBDs-C, -D, and -E retrieve a 20-nt G-quadruplex forming sequence. Binding studies show that RPA-DE binds preferentially to the G-quadruplex DNA, a unique preference not observed with other RPA constructs. Circular dichroism experiments show that RPA-CDE-core can unfold the G-quadruplex while RPA-DE stabilizes it. Binding studies show that RPA-C binds pyrimidine- and purine-rich sequences similarly. This difference between RPA-C and RPA-DE binding was also indicated by the inability of RPA-CDE-core to unfold an oligonucleotide containing a TC-region 5′ to the G-quadruplex. Molecular modeling studies of RPA-DE and telomere-binding proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA-DE and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties

A study of 7-deaza-2′-deoxyguanosine–2′-deoxycytidine base pairing in DNA

The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson–Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C–H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization

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Topoisomerase 1 (Top1) enzymes regulate DNA superhelicity by forming covalent cleavage complexes that undergo controlled rotation. Substitution of nucleoside analogs at the +1 position of the DNA duplex relative to the Top1 cleavage site inhibits DNA religation. The reduced efficiency for Top1-mediated religation contributes to the anticancer activity of widely used anticancer drugs including fluoropyrimidines and gemcitabine. In the present study, we report that mismatched base pairs at the +1 position destabilize the duplex DNA components for a model Top1 cleavage complex formation even though one duplex component does not directly include a mismatched base pair. Molecular dynamics simulations reveal G-dU and G-FdU mismatched base pairs, but not a G-T mismatched base pair, increase flexibility at the Top1 cleavage site, and affect coupling between the regions required for the religation reaction to occur. These results demonstrate that substitution of dT analogs into the +1 position of the non-scissile strand alters the stability and flexibility of DNA contributing to the reduced efficiency for Top1-mediated DNA religation. These effects are inherent in the DNA duplex and do not require formation of the Top1:DNA complex. These results provide a biophysical rationale for the inhibition of Top1-mediated DNA religation by nucleotide analog substitution

Characterization of DNA with an 8-oxoguanine modification

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2′-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2–8 kcal mol−1 over a 16–116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5′ of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding