Parallel G-quadruplexes formed by guanine-rich microsatellite repeats inhibit human topoisomerase I

Abstract

Using UV and CD spectroscopy, we studied the thermodynamic stability and folding topology of G-quadruplexes (G4), formed by G-rich fragments in human microsatellites that differ in the number of guanosines within the repeating unit. The oligonucleotides d(GGGT)4 and d(GGT)4 were shown to form propeller-type parallel-stranded intramolecular G-quadruplexes. The G4 melting temperature is dramatically decreased (by more than 45°C) in the transition from the tri-G-tetrad to the bi-G-tetrad structure. d(GT)n-repeats do not form perfect G-quadruplexes (one-G-tetrad); folded G4-like conformation is not stable at room temperature and is not stabilized by monovalent metal ions. The minimum concentration of K+ that promotes quadruplex folding of d(GGT)4 was found to depend on the supporting Na+ concentration. It was demonstrated for the first time that the complementary regions flanking G4-motifs (as in d(CACTGG-CC-(GGGT)4-TA-CCAGTG)) cannot form a double helix in the case of a parallel G4 due to the steric remoteness, but instead destabilize the structure. Additionally, we investigated the effect of the described oligonucleotides on the activity of topoisomerase I, one of the key cell enzymes, with a focus on the relationship between the stability of the formed quadruplexes and the inhibition degree of the enzyme. The most active inhibitor with IC50 = 0.08 µM was the oligonucleotide d(CACTGG-CC-(GGGT)4-TA-CCAGTG), whose flanking G4-motif sequences reduced the extreme stability of G-quadruplex formed by d(GGGT)4.

The Functional Consequences of Eukaryotic Topoisomerase 1 Interaction with G-Quadruplex DNA

“…Aptamers targeting proteins or other biologically relevant molecules have been selected by screening libraries of DNA or RNA oligos and further studied for the potential therapeutic application. Since it was first discovered that Top1 binds and is inhibited by G4-forming oligos [37,38], other similar results have been reported [39,48,49]. First, using purified Calf Thymus Top1, Shuai et al characterized fourteen different guanine-rich oligos capable of forming either G-quadruplex or quadruplex-duplex hybrid and showed that all of the oligos act as competitive inhibitors of Top1 catalysis [39].…”

Assembly of a G-Quadruplex Repair Complex by the FANCJ DNA Helicase and the REV1 Polymerase

“…To test this, we monitored the binding of the peptide and a quadruplex formed by (GGGT) 4 with 8-oxoguanine at the corresponding 8oxo1 and 8oxo5 positions. This sequence was chosen because the single thymidine spacers produced a compact parallel G4 with high thermal stability (T m =~85 • C) [58]. FANCJ AKKQ bound to (GGGT) 4 with lowered affinity K = 2.7 ± 0.4 × 10 5 M −1 compared to (TTAGGG) 4 .…”

Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

“…The type of loops connecting G tracts, their length, and base sequence represent such factors [40 42]. The two or three tetrad G4 with three single nucleotide loops has parallel topology and no other, and all the loops are of propeller type [43,44]. In the presence of K + , the struc ture with parallel orientation of all the strands is charac teristic for the quadruplex with two single nucleotide loops regardless of the length and base sequence of the third loop [26,42,45].…”

“…The 3D structure of an aptamer with similar base sequence was investigated by X ray crystallography [112]. Formation of the duplex domain was not observed when the mutually complementary sequences flanked the parallel G4 because in this case the complementary frag ments were separated in space; their existence only desta bilized the G4 structure [44]. Fig.…”

“…Data are available on the effect of G4 on translo cation of the telomerase catalytic subunit (TERT) from the nucleus to mitochondria, which plays an important role in cellular apoptosis induced by the G quadruplex binding ligands [309]. It was established that G4s of different topol ogy could inhibit topoisomerase I mediated nicking of double stranded DNA [310,311], thus affecting the degree of DNA supercoiling. The specialized low fidelity DNA polymerases hPol η and hPol ε from the Y and B families, respectively, which are capable of replicative bypassing base damaged sites, retain various activity towards G4 containing substrates depending on the degree of specific binding with G quadruplexes [279].…”