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.

Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase β on the APE1–DNA interaction

“…According to the "passing-the-baton" model of BER, DNA intermediates in BER are processed and then passed from one enzyme to another in a coordinated fashion [44][45][46]. Step-by-step coordination of BER events is thought to be facilitated by multiple protein-protein interactions comprising BER enzymes and accessory proteins [43,[47][48][49][50][51]. Therefore, investigating not only protein-DNA but also protein-protein interactions is required for a complete understanding of the BER mechanism.…”

“…Besides, APE1 is reported to stimulate the dRP lyase activity of Polβ [70,71]. In turn, APE1 endonuclease activity is enhanced by some DNA glycosylases [50], Polβ [50,72], and XRCC1 [37,50,72]. The mutual enhancement of each other's enzymatic activities by APE1 and aforementioned proteins can materialize due to either direct [73,74] or DNA-mediated [51] protein-protein interactions.…”

Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker

“…The activation of the DNAzyme walker involves both DNA glycosylase activity and the cleavage of the AP site. Thus, the activities of UDG and SMUG1, along with the cleavage of the AP site by APE1 lyase activity, are necessary for the generation of fluorescence by the DNAzyme walker. , APE1 is known to coordinate with DNA glycosylases and stimulate glycosylase activity. ,− Furthermore, glycosylases within cancer cells likely have highly varying activities because of the plethora of proteins and pathways that interact with the glycosylases. Therefore, the DNAzyme-mediated responses reported here should be understood to result not only from base excision but also from a dynamic coordination between the glycosylases, APE1, and other factors in cancer cells.…”

Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients

“…The downregulation of NEIL1 was previously reported to be associated with increased oxidation DNA damage [ 23 ]. APEX1 is the dominant AP endonuclease in human cells to incise the AP site after the removal of the damaged base after excision has taken place with a monofunctional DNA glycosylase [ 24 ].…”

“…As mentioned before, the value of using placental tissues is the non-invasive collection of the biomatrix and the ability to study molecular markers early in life in relation to exposure or disease. Previous studies have assessed DNA repair activity in placental tissue in an indirect way via the study of gene expression [ 23 , 24 , 25 ] or protein expression [ 47 ], and genetic variants [ 24 , 26 , 27 ], However, studying gene variants or expression does not necessarily give any indication of enzyme activity. The lack of correspondence between DNA repair gene expression and enzyme activity has been discussed before [ 46 ].…”