Substrate specificity of human apurinic/apyrimidinic endonuclease APE1 in the nucleotide incision repair pathway

Abstract

Human apurinic/apyrimidinic (AP) endonuclease APE1 catalyses the hydrolysis of phosphodiester bonds on the 5′ side of an AP-site (in the base excision repair pathway) and of some damaged nucleotides (in the nucleotide incision repair pathway). The range of substrate specificity includes structurally unrelated damaged nucleotides. Here, to examine the mechanism of broad substrate specificity of APE1, we performed pulsed electron–electron double resonance (PELDOR) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer (FRET) detection of DNA conformational changes during DNA binding and lesion recognition. Equilibrium PELDOR and kinetic FRET data revealed that DNA binding by APE1 leads to noticeable damage-dependent bending of a DNA duplex. Molecular dynamics simulations showed that the damaged nucleotide is everted from the DNA helix and placed into the enzyme’s binding pocket, which is formed by Asn-174, Asn-212, Asn-229, Ala-230, Phe-266 and Trp-280. Nevertheless, no damage-specific contacts were detected between these amino acid residues in the active site of the enzyme and model damaged substrates containing 1,N6-ethenoadenosine, α-adenosine, 5,6-dihydrouridine or F-site. These data suggest that the substrate specificity of APE1 is controlled by the ability of a damaged nucleotide to flip out from the DNA duplex in response to an enzyme-induced DNA distortion.

The Role of Active-Site Plasticity in Damaged-Nucleotide Recognition by Human Apurinic/Apyrimidinic Endonuclease APE1

“…PAGE analysis of product accumulation in the course of the interaction of APE1 with every X-substrate (X = αA, DHU, F-site, or εA as damage) was performed first. Direct registration of product formation helped to rank the efficacy of DNA cleavage as follows: F-site > DHU >> αA > εA, in line with other studies [ 25 , 30 , 33 ].…”

“… Changes in the FRET signal during the interaction of APE1 with each FRET-X-substrate in the absence “−” or presence “+” of 5.0 mM Mg 2+ . FRET traces of the interaction of APE1 with the FRET-F- and FRET-DHU-substrates in the absence of Mg 2+ have been reported earlier [ 25 ]. …”

“…In our previous study [ 25 ], an analysis of conformational dynamics during an interaction of this enzyme with DNA containing 1,N6-ethenoadenosine (εA), α-adenosine (αA), 5,6-dihydrouridine (DHU), or an F-site revealed that the DNA distortions induced by enzyme binding and initial-complex formation depend on the nature of the damaged nucleotide. It has been suggested that the key factor responsible for the substrate specificity of APE1 is the ability of a damaged nucleotide to get everted from a double helix and to get inserted into the damaged-nucleotide binding pocket.…”

Structural and Functional Characterization of a Unique AP Endonuclease From Deinococcus radiodurans

“…APE1 catalyzes the cleavage of AP- and 5,6-dihydrouridine (DHU)-containing substrates in the same active site ( Felix and Silveira, 2011 ). Moreover, molecular dynamics simulations showed that the broad substrate specificity of APE1 is due to DNA distortion induced by the enzyme, which flips damaged nucleotides into the active site pocket ( Kuznetsova et al, 2018 ). DrXth lacks NIR activity, similar to most prokaryotic ExoIII-like enzymes (e.g., EcXth and MtbXthA).…”

Effect of the Substrate Structure and Metal Ions on the Hydrolysis of Undamaged RNA by Human AP Endonuclease APE1

“…Therefore, human AP endonuclease is versatile in terms of the functions it plays in the cell. Structural data, kinetic studies and a mutation analysis have made it possible to identify the key stages of interaction between APE1 and damaged DNA harboring the AP site [ 15 , 16 , 17 ], as well as some damaged [ 18 ] and undamaged [ 7 ] nucleotides. Kuznetsova et al have suggested a mechanism for the broad substrate specificity of AP endonuclease exhibited upon its interaction with DNA [ 18 ].…”