Quantitative Analysis of Stall of Replicating DNA Polymerase by G-Quadruplex Formation
The formation of guanine-quadruplexes (G4s) in genomic DNA and RNA inhibits replication, transcription, and translation. Although several regions of the human genome exhibit G4-forming potential, the stability and topology of the G4s formed vary depending
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Introduction Guanine-quadruplexes (G4s) are one of the non-canonical structures of nucleic acids. G4s consist of stacked G-tetrad structures from four strands of G-tracts. Recently, it has been identified that G4 structures form in cells and regulate gene expression processes such as replication, transcription, and translation [1–6]. The stability of G4s depends on the sequence and surrounding conditions such as ions and cosolutes. As the cellular environment is a condition of molecular crowding that is occupied by a high concentration of molecules including metabolites and macromolecules [7], the effect of G4s on gene expression should be considered from a physicochemical viewpoint. In addition, there are a variety of different topologies of G4 structures such as antiparallel, mixed, and parallel type (Fig. 1). Furthermore, the loop regions between each G-tract can be important in forming and stabilizing the G4 structures. These different structures can be a target of G4 ligands that control G4 formation, resulting in disease due to the unnatural formation of G4s. More importantly, G4 topology can change depending on the crowding conditions [8]. Therefore, it is crucial to quantitatively analyze the effect of G4 stability and structures on
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_15, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Shuntaro Takahashi and Naoki Sugimoto
G-quadruplex (A)
(B)
I-motif (C)
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(D)
Hairpin (E)
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5’
3’ 5’ 5’
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Fig. 1 Schematic illustrations of topologies of an (a) antiparallel G4, (b) mixed G4, (c) parallel G4, (d) i-motif, and (e) hairpin. The base pairs are represented by blue rectangles. Other bases in the loop region are not indicated in the illustrations
gene expression in molecular crowding conditions. Here, we demonstrate a quantitative study of topology-dependent replication (called QSTR here) to analyze the replication reaction along G4-forming template DNA in various molecular environments [4]. First, the stability (ΔG ) of G4 is determined by increasing the temperature to melt the DNA structure. The process of melting is assayed by spectroscopic techniques upon changes in temperature. Second, the replication assays are carried out using template DNAs having G4 structures in the presence of DNA polymerase (DNAP) and its substrates. The rate constants of replication (ks) to overcome the G4 structure are determined by the time course of replication products, because G4 structures on the template DNA interrupt the processivity of DNAPs. Finally, the stability of G4 and rate of replication are correlated by an lnks vs. ‑ΔG plot. Since ‑RT lnks indicates the activation energy (ΔG{) of replication (R is the gas constant, and T is temperature), we can understand the mechanism of replication inhibition by comparing the ΔG{ value of replication of G4s. QSTR can be a basic protocol to evaluate mole
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