The role of active-site amino acid residues in the cleavage of DNA and RNA substrates by human apurinic/apyrimidinic endonuclease APE1

The Enigma of Substrate Recognition and Catalytic Efficiency of APE1-Like Enzymes

“…To follow the conformational changes in DNA by detecting changes in the FRET signal, we used the FRET-F-duplex with FAM attached to the 5 end of the damaged strand and a BHQ1 quencher residue attached to the 5 end of the complementary strand. It should be noted that FRET detection has been previously utilized for the analysis of DNA cleavage activity of hAPE1 (Alekseeva et al, 2019a(Alekseeva et al, , 2020Kladova et al, 2020). As shown in Figure 3C, FRET detection revealed a significant increase in the signal that corresponds to catalytic cleavage of the F-site for all the tested AP endonucleases.…”

“…Accordingly, it is always useful to study the same reaction by following the changes in several types of fluorescence. Previously, a significant amount of data has been obtained on the kinetics of hAPE1 interacting with different substrates by means of the fluorescence of Trp residues in the enzyme (Timofeyeva et al, 2009(Timofeyeva et al, , 2011Kuznetsova et al, 2018b;Alekseeva et al, 2019b) and also DNA substrates that contain aPu as a fluorescent nucleotide analog (Kanazhevskaya et al, 2012;Kuznetsova et al, 2014Kuznetsova et al, , 2018a or an emitter/quencher pair of dyes to measure FRET (Miroshnikova et al, 2016b;Alekseeva et al, 2019aAlekseeva et al, , 2020. It was tempting to use the data known for hAPE1 as a reference of all APE1-like enzymes tested in the present study.…”

“…A major human AP endonuclease, human APE1 (hAPE1) is one of the most studied AP endonucleases. Indeed, multiple structural data (Gorman et al, 1997;Mol et al, 2000a,b;Beernink et al, 2001;Manvilla et al, 2013;Tsutakawa et al, 2013;Freudenthal et al, 2015), kinetic studies (Timofeyeva et al, 2009;Miroshnikova et al, 2016a,b;Alekseeva et al, 2019a), and a mutational analysis (Erzberger and Wilson, 1999;Alekseeva et al, 2020) have made it possible to identify the key stages of the interaction of hAPE1 with a damaged DNA harboring an AP site or with damaged (Timofeyeva et al, 2011;Timofeyeva and Fedorova, 2016;Kuznetsova et al, 2018b;Bulygin et al, 2020) or undamaged (Kuznetsova et al, 2018a(Kuznetsova et al, , 2020 nucleotides.…”

“…Another set of amino acid residues (Asp70, Glu96, Tyr171, Asp210, Asn212, Asp308, and His309) is responsible for the coordination of the cofactor Mg 2+ and scissile phosphate group of the damaged nucleotide. The role of Mg 2+ ions in the DNA binding and catalytic mechanism is still debated (Gorman et al, 1997;Masuda et al, 1998;Erzberger and Wilson, 1999;Mol et al, 2000b;Beernink et al, 2001;Oezguen et al, 2007;Lipton et al, 2008;Manvilla et al, 2013;He et al, 2014;Miroshnikova et al, 2016b;Alekseeva et al, 2020).…”

Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity

“…Mutant forms of APE1 containing the substitutions Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A were purified in the same way, except that the E. coli cells were transformed with expression vectors carrying the corresponding nucleotide substitutions, as described previously [17]. Mutant forms of APE1 containing the substitutions Y171F, R177F, and R181A were obtained for the first time and the corresponding mutations were introduced into the enzyme gene by site-directed mutagenesis (Quik-Change XL; Stratagene, United States).…”