Modulation of the Apurinic/Apyrimidinic Endonuclease Activity of Human APE1 and of Its Natural Polymorphic Variants by Base Excision Repair Proteins

Abstract

Human apurinic/apyrimidinic endonuclease 1 (APE1) is known to be a critical player of the base excision repair (BER) pathway. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that these proteins interact with APE1 either at upstream or downstream steps of BER. Therefore, we may propose that even a minor disturbance of protein-protein interactions on the DNA template reduces coordination and repair efficiency. Here, the ability of various human DNA repair enzymes (such as DNA glycosylases OGG1, UNG2, and AAG; DNA polymerase Polβ; or accessory proteins XRCC1 and PCNA) to influence the activity of wild-type (WT) APE1 and its seven natural polymorphic variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S) was tested. Förster resonance energy transfer-based kinetic analysis of abasic site cleavage in a model DNA substrate was conducted to detect the effects of interacting proteins on the activity of WT APE1 and its single-nucleotide polymorphism (SNP) variants. The results revealed that WT APE1 activity was stimulated by almost all tested DNA repair proteins. For the SNP variants, the matters were more complicated. Analysis of two SNP variants, R237A and G241R, suggested that a positive charge in this area of the APE1 surface impairs the protein-protein interactions. In contrast, variant R221C (where the affected residue is located near the DNA-binding site) showed permanently lower activation relative to WT APE1, whereas neighboring SNP N222H did not cause a noticeable difference as compared to WT APE1. Buried substitution P311S had an inconsistent effect, whereas each substitution at the DNA-binding site, M270T and R274Q, resulted in the lowest stimulation by BER proteins. Protein-protein molecular docking was performed between repair proteins to identify amino acid residues involved in their interactions. The data uncovered differences in the effects of BER proteins on APE1, indicating an important role of protein-protein interactions in the coordination of the repair pathway.

Keywords: AP endonuclease; DNA repair; coordination of DNA repair process; protein–protein interaction; single-nucleotide polymorphism.

Figure 1

Spatial location of the amino acid residues affected by the SNPs of APE1 (PDB ID 1DE8).

Figure 2

Interaction of WT APE1 or its SNP variants with the FRET-labeled substrate. The FRET signal changes characterize the activity of WT APE1 and SNP variants without any effectors (A) or in the presence of AAG (B), OGG1 (C), UNG2 (D), Polβ (E), PCNA (F), or XRCC1 (G).

Figure 3

A comparison of stimulation coefficients f of BER proteins between APE1 WT and its SNP variants. The stimulation coefficient is computed according to Equation (2). [WT APE1 or SNP variant] = 10 nM, [FRET-F substrate] = 1.0 μM, [effector protein (AAG, OGG1, UNG2, Polβ, PCNA, or XRCC1)] = 1.0 μM.

Figure 4

Protein–protein docked models of APE1 complexes with BER enzymes: OGG1 (A), AAG (B), UNG2 (C), Polβ (D), or PCNA (E). The PDB structures employed for the docking are 1DE8 for APE1, 1EBM for OGG1, 1F4R for AAG, 1EMH for UNG2, 4PHD for Polβ, and 6FCM for PCNA.

Figure 4

Protein–protein docked models of APE1 complexes with BER enzymes: OGG1 (A), AAG (B), UNG2 (C), Polβ (D), or PCNA (E). The PDB structures employed for the docking are 1DE8 for APE1, 1EBM for OGG1, 1F4R for AAG, 1EMH for UNG2, 4PHD for Polβ, and 6FCM for PCNA.

Figure 5

Possible amino acid interfaces participating in the protein–protein interactions around the area of a substituted amino acid residue in the SNP variants of APE1 as documented for OGG1 (A), AAG (B), UNG2 (C), Polβ (D), and PCNA (E). Surface amino acid residues of APE1 (Arg221, Asn222, Arg237, and Gly241) and amino acid residues of the effector proteins, if any, that were found near the SNP-induced substitutions in APE1 are illustrated in the transparent view of the electrostatic potential.