Binding of AP Endonuclease-1 to G-Quadruplex DNA Depends on the N-Terminal Domain, Mg²⁺, and Ionic Strength

Abstract

The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the noncanonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1’s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+ and Mg2+ and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+ found that dsDNA and G4 substrates required the cation for both binding and catalysis, in which the G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ ions showed that F-containing dsDNA was bound and cleaved by APE1, whereas the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using Δ33 or Δ61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding, and catalysis is nearly abolished without the N-terminus. With this knowledge, we found that APE1 could remodel the F-containing VEGF promoter dsDNA → G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.

Cysteine Oxidation to Sulfenic Acid in APE1 Aids G-Quadruplex Binding While Compromising DNA Repair

“…To observe the effect of the multivalent cation presence in APE1 binding, the assays were repeated in the presence of 10 mM Sm 3+ as a substitute for Mg 2+ with the purpose of preventing any catalytic activity on the substrates but not to impact effects on the binding . Our prior work found the binding between catalytically inactivated APE1 and a G4 was Mg 2+ -dependent . Therefore, the discussion here will focus on the Sm 3+ studies, but for completeness of the study, binding without Mg 2+ was analyzed and is provided in Figure S10.…”

“…To understand the role of cysteine oxidation to sulfenic acids in APE1 in influencing DNA binding, fluorescence anisotropy binding assays were conducted following a method previously reported . The binding studies were conducted using the sequences shown in Table .…”

“…Initially, all binding assays were conducted without Mg 2+ and in the presence of 1 mM EDTA to prevent any cleavage activity because this divalent metal is required for strand scission . To observe the effect of the multivalent cation presence in APE1 binding, the assays were repeated in the presence of 10 mM Sm 3+ as a substitute for Mg 2+ with the purpose of preventing any catalytic activity on the substrates but not to impact effects on the binding .…”

Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression

“…The AP site and BER machinery destabilize the normal double helix structure and lower the energy barrier for forming G-quadruplexes in some sequences [ 31 ]. The AP endonuclease of Ape1 is highly attenuated at abasic sites in certain parts of a G-quadruplex structure, compared with its activity on double-strand DNA [ 32 ]. In such cases, rather than incising the phosphodiester bond, Ape1 now helps stabilize these G-quadruplexes.…”

Fluorophore‐mediated photooxidation of the guanine heterocycle

“…Fluorophores attached to DNA or RNA oligomers have enabled monitoring their migration during gel electrophoresis experiments [1] and the tracking of these polymers in single-molecule experiments. [2,3] Additionally, the binding interactions between DNA or RNA with other nucleic acids, [4] proteins, [5] or ligands, [6] as well as in sequencing-by-synthesis experiments, can be followed. [7] There exist many fluorophores tailored for various applications in which fluorescein, rhodamine, BODIPY, and cyanine3 (Cy3) or variants thereof are used with some frequency in nucleic acid visualization (Figure 1).…”