molecules

Article Kinetic Features of 30-50 Exonuclease Activity of Human AP- APE1

Alexandra A. Kuznetsova 1, Olga S. Fedorova 1,2,* ID and Nikita A. Kuznetsov 1,2,* ID

1 Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia; [email protected] 2 Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia * Correspondence: [email protected] (O.S.F.); [email protected] (N.A.K.); Tel.: +7-383-363-5175 (O.S.F.); +7-383-363-5174 (N.A.K.); Fax: +7-383-363-5153 (O.S.F. & N.A.K.)  Received: 19 July 2018; Accepted: 16 August 2018; Published: 21 August 2018 

Abstract: Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 50 to the damage. In addition to the AP-endonuclease activity, APE1 possesses 30-50 exonuclease activity, which presumably is responsible for cleaning up nonconventional 30 ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 30-end removal in the 30-50 exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 50 dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 30 end of the short oligonucleotide. The impact of the 30-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 30-50 exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 30 nucleotide removal by APE1 in the 30-50 exonuclease process is the release of the detached nucleotide from the ’s .

Keywords: AP-endonuclease; DNA repair; exonuclease activity; pre-steady-state kinetics

1. Introduction Human apurinic/apyrimidinic endonuclease 1 (APE1), also known as a redox factor 1 (Ref-1) is a multifunctional enzyme [1]. The primary physiological function of APE1 is the incision of the phosphodiester bond immediately 50 to an apurinic/apyrimidinic (AP) site, which generates a single-strand break with 50-deoxyribose phosphate and 30-hydroxyl ends (Figure1)[2–4]. APE1 possesses some minor activities: 30-50 exonuclease, 30-phosphodiesterase, 30-phosphatase, and RNase H [5–8]. In addition to these activities, APE1 can recognize and convert some damaged or modified such as 5,6-dihydrouridine [9], α-anomers [10], etheno derivatives [11,12], bulky photoadducts [13], benzene derivatives [14], and 20-deoxyuridine [15]. This type of APE1 activity was named as the “nucleotide incision repair” (NIR) activity [16]. The heterogeneity of substrate specificity of the enzyme could be elucidated by structural data. Nonetheless, the currently available structural data mainly represent different variants of the free enzyme [17–19] and its complexes with nicked or abasic DNA [20–22]. Analysis of these data has shown that, for endonuclease activity, specific contacts in the complex APE1·DNA are formed, which result in the change of DNA conformation including eversion of an AP-site from the double helix. At the same time, the binding to DNA leads to slight structural rearrangements in the APE1 molecule itself. It has also been shown that residues of APE1 interact mainly with only one DNA chain, which contains an AP-site by forming

Molecules 2018, 23, 2101; doi:10.3390/molecules23092101 www.mdpi.com/journal/molecules Molecules 2018, 23, 2101 2 of 14

Molecules 2018, 23, x 2 of 14 Moleculeshydrogen 2018 bonds, 23, x and electrostatic contacts between DNA backbone phosphates and amino acid2 sideof 14 hydrogen bonds and electrostatic contacts between DNA backbone phosphates and amino acid side groups or amide groups of peptide bonds. Recently, a set of APE1-DNA structural snapshots was hydrogengroups or bonds amide and groups electrostatic of peptide contacts bonds. between Recent ly,DNA a set backbone of APE1-DNA phosphates structural and amino snapshots acid wasside reported revealing that APE1 removes 30 mismatches and 30-phosphoglycolate by placing the 30 group groupsreported or revealingamide groups that APE1 of peptide removes bonds. 3′ mismatches Recently, and a set 3′-phosphoglycolate of APE1-DNA structural by placing snapshots the 3′ group was within the intra-helical DNA cavity via a non-base-flipping mechanism [23]. It has been suggested reportedwithin the revealing intra-helical that APE1 DNA removes cavity via 3′ mismatches a non-base-flipping and 3′-phosphoglycolate mechanism [23]. byIt hasplacing been the suggested 3′ group that the hydrophobic pocket, which is composed of Phe-266 and Trp-280, plays a role in the substrate withinthat the the hydrophobic intra-helical pocket, DNA cavitywhich isvia composed a non-base-flipping of Phe-266 and mechanism Trp-280, plays[23]. Ita rolehas beenin the suggested substrate specificity [23,24]. The catalytic reaction is initiated by a nucleophilic attack of the oxygen atom of an thatspecificity the hydrophobic [23,24]. The pocket, catalytic which reaction is composed is initiate ofd Phe-266by2+ a nucleophilic and Trp-280, attack plays of the a0 role oxygen in the atom substrate of an H2O molecule coordinated directly or through an Mg ion by Asp-210 on the 5 -phosphate group of specificityH2O molecule [23,24]. coordinated The catalytic directly reaction or through is initiate and Mg by 2+a ionnucleophilic by Asp-210 attack on the of 5the′-phosphate oxygen atom group of ofan the AP-site [20,22]. In alternative catalytic mechanisms [25,26], the role of the nucleophilic base plays Hthe2O moleculeAP-site [20,22]. coordinated In alternative directly catalytic or through mechanisms an Mg2+ [25,26],ion by Asp-210 the role ofon the the nucleophilic 5′-phosphate base group plays of the His-309 residue or the phenolate form of the Tyr-171 residue. thethe AP-site His-309 [20,22]. residue In or alternative the phenol catalyticate form mechanisms of the Tyr-171 [25,26], residue. the role of the nucleophilic base plays the His-309 residue or the phenolate form of the Tyr-171 residue.

Figure 1. Types of catalytic activity for APE1. Figure 1. Types of catalytic activity for APE1. Figure 1. Types of catalytic activity for APE1. In our earlier studies, kinetic analysis of conformational changes of the enzyme and of a DNA moleculesIn our earlierhas been studies, performed kinetic on analysis the endonuclease of conformational process catalyzed changes of by the APE1 enzyme [27–29]. and For of a DNA DNA In our earlier studies, kinetic analysis of conformational changes of the enzyme and of a DNA moleculessubstrateshas containing been performed an AP-site on or the its endonucleasesynthetic analog process 2-oxymethyl-3-oxy-tetrahydrofuran catalyzed by APE1 [27–29]. For (F-site), DNA molecules has been performed on the endonuclease process catalyzed by APE1 [27–29]. For DNA substratesthe kinetic containing scheme containing an AP-site two or reversible its synthetic step analogs of DNA-substrate 2-oxymethyl-3-oxy-tetrahydrofuran binding has been determined (F-site), substrates containing an AP-site or its synthetic analog 2-oxymethyl-3-oxy-tetrahydrofuran (F-site), the(Scheme kinetic 1) scheme [30,31]. containing It was hypothes two reversibleized that steps the first of DNA-substrate binding step represents binding has the been formation determined of a the kinetic scheme containing two reversible steps of DNA-substrate binding has been determined (Schemenonspecific1)[ 30complex,31]. It of was APE1 hypothesized with DNA: that(E·S) the1. In first the bindingcourse of step the representssecond step, the when formation the (E·S) of2 a (Scheme 1) [30,31]. It was hypothesized that the first binding step represents the formation of a nonspecificcomplex is complexproduced, of the APE1 amino with acid DNA: residues (E·S) and1. In a theMg course2+ ion in of the the active second site step, of the when enzyme the (Eform·S)2 nonspecific complex of APE1 with DNA: (E·S)1. In the course of the second step, when the (E·S)2 complexspecific contacts is produced, with the5′ and amino 3′ phosphates acid residues and andribose a Mgresidues2+ ion of in AP-DNA. the active During site of this the enzymestep, the formAP- complex is produced, the amino acid residues and a Mg2+ ion in the active site of the enzyme form specificsite is everted contacts to withthe active 50 and site 30 phosphatesof the enzyme and so ribosethat a catalytically residues of AP-DNA.active conformation During this of step,APE1the is specific contacts with 5′ and 3′ phosphates and ribose residues of AP-DNA. During this step, the AP- AP-sitegenerated. is everted After tothat, the the active irreversible site of the chemical enzyme st soep that of ahydrolysis catalytically of the active phosphodiester conformation bond of APE1 5′ to is site is everted to the active site of the enzyme so that a catalytically active conformation of APE1 is generated.the AP-site After proceeds. that, the The irreversible final, fourth chemical step is stepthe reversible of hydrolysis release of the of phosphodiesterproduct P, which bond contains 50 to the a generated. After that, the irreversible chemical step of hydrolysis of the phosphodiester bond 5′ to AP-sitenick in proceeds.one DNA Thechain final, from fourth the complex step is thewith reversible the enzyme release E·P. of product P, which contains a nick in the AP-site proceeds. The final, fourth step is the reversible release of product P, which contains a one DNA chain from the complex with the enzyme E·P. nick in one DNA chain from the complex with the enzyme E·P.

Scheme 1. The kinetic mechanism of the interaction between APE1 and DNA containing an abasic

site. Е is the enzyme, S is a DNA substrate, (E·S)1 and (E·S)2 are complexes of the enzyme with the Scheme 1. The kinetic mechanism of the interaction between APE1 and DNA containing an abasic Schemesubstrate, 1. TheР is a kinetic product mechanism of substrate of conversion, the interaction E·P is between a complex APE1 of the and enzyme DNA containing with the product, an abasic ki site.and Е k −isi denotethe enzyme, rate constants S is a DNA of di substrate,rect and reverse (E·S)1 reactionsand (E·S) 2( iare = 1 complexesor 2) of reversible of the enzymesteps, k3 withis a rate the site. E is the enzyme, S is a DNA substrate, (E·S)1 and (E·S)2 are complexes of the enzyme with the substrate,constant Рof isthe a productcatalytic ofreaction, substrate and conversion, Kp is the equilibrium E·P is a complex dissociation of the constant enzyme for with the the Е·Р product, complex. k i substrate, P is a product of substrate conversion, E·P is a complex of the enzyme with the product, ki and k−i denote rate constants of direct and reverse reactions (i = 1 or 2) of reversible steps, k3 is a rate and k−i denote rate constants of direct and reverse reactions (i = 1 or 2) of reversible steps, k3 is a rate constantIn spite of of the intensive catalytic studiesreaction, regarding and Kp is the the equilibrium features of dissociation the major endonuclconstant forease the activity Е·Р complex. of APE1, constant of the catalytic reaction, and Kp is the equilibrium dissociation constant for the E·P complex. the mechanisms of other activities of this enzyme toward DNA substrates with significantly different structuresIn spite are of intensivestill insufficiently studies regardingexamined. the It was featur shownes of earlierthe major that, endonucl under conditionsease activity optimal of APE1, for theAP mechanisms endonucleaseIn spite of intensive of activity,other activities studies the 3′-5 regarding ′of exonuclease this enzyme the activity features toward is of DNAsix the to seven majorsubstrates orders endonuclease with of magnitude significantly activity lower different of APE1, than structuresthethe mechanisms endonuclease are still of activityinsufficiently other activities [32]. Ther examined. ofefore, this enzyme in It the was present toward shown work DNA earlier we substrates werethat, focusedunder with conditions on significantly the comparison optimal different forof APstructuresvarious endonuclease DNA are still substrates activity, insufficiently thein 3the′-5 examined. ′ courseexonuclease of It only was activity shown3′-5′ isexonuclease earliersix to seven that, processing. underorders conditions of magnitude For this optimal purpose, lower for thanAP a thecomparative endonuclease kinetic activity analysis [32]. of Ther theefore, conformational in the present changes work of we model were DNAfocused substrates on the comparison in the course of various DNA substrates in the course of only 3′-5′ exonuclease processing. For this purpose, a comparative kinetic analysis of the conformational changes of model DNA substrates in the course

Molecules 2018, 23, 2101 3 of 14 endonuclease activity, the 30-50 exonuclease activity is six to seven orders of magnitude lower than the endonuclease activity [32]. Therefore, in the present work we were focused on the comparison 0 0 ofMolecules various 2018 DNA, 23, x substrates in the course of only 3 -5 exonuclease processing. For this purpose,3 of 14 a comparative kinetic analysis of the conformational changes of model DNA substrates in the course of theirtheir recognitionrecognition andand 330′-5-50′ exonucleaseexonuclease cleavage byby APE1APE1 waswas performed.performed. By the stopped-flowstopped-flow fluorescencefluorescence technique,technique, changes in the fluorescencefluorescence of 2-aminopurine residues introduced at various positionspositions ofof thethe DNADNA substratesubstrate werewere recorded.recorded. The effects ofof undamagedundamaged andand damageddamaged 330′-end-end nucleotides or stabilitystability ofof thethe 330′-end-end pairpair onon thethe 330′-5-50′ exonucleaseexonuclease activityactivity werewere estimated.estimated. Taken together,together, the obtained obtained data data allowed allowed us us to to elucidat elucidatee kinetic kinetic features features of ofthe the mechanism mechanism underlying underlying the the3′-5′ 3 0exonuclease-50 exonuclease activity activity of APE1 of APE1 and and to toconclude conclude that that the the rate-limiting rate-limiting step step is is the the release release of the detached nucleotide fromfrom thethe activeactive sitesite ofof thethe enzyme.enzyme.

2. Results and Discussion 0 Exonuclease activity activity of of APE1 APE1 is iseffective effective toward toward duplexes duplexes containing containing gaps gaps or 5′-dangling or 5 -dangling ends 0 0 ends[32–34]. [32 –Therefore,34]. Therefore, for the for kinetic the kineticanalysis analysis of the 3′ of-5′ theexonuclease 3 -5 exonuclease reaction, reaction,the duplexes the duplexesof 15 and 28 of 0 15nucleotides and 28 nucleotides (nt) with (nt) a 5 with′-dangling a 5 -dangling end served end served as model as model DNA DNA substrates substrates (Figure (Figure 2).2). The The 15 ntnt 0 oligonucleotides contained the aPu residue at position 1, 2, 4, or 6 from thethe 33′ end in thethe duplexesduplexes j Exo-aPu j/T (j = 1, 2, 4, and 6). The fluorescence fluorescence of aPu is significantlysignificantly quenched when this base is incorporatedincorporated intointo single-strandedsingle-stranded oror double-strandeddouble-stranded DNADNA [[35–37].35–37]. This property of aPu enabled us toto followfollow thethe ratesrates ofof itsits removalremoval fromfrom thethe differentdifferent positionspositions inin aa DNADNA chain.chain.

Figure 2. Schematic structures of DNA duplexes Exo-aPuj/T (a), Exo-aPu1/N (b), and Exo-aPu2-X/N Figure 2. Schematic structures of DNA duplexes Exo-aPuj/T (a), Exo-aPu1/N (b), and Exo-aPu2-X/N (c) used as substrates and ligands of APE1. (c) used as substrates and ligands of APE1. Moreover, using DNA substrates containing mismatched or damaged 3′ nucleotides allowed us 0 to estimateMoreover, the influence using DNA of th substratese opposite-strand containing nucleotide mismatched (Exo-aPu or damaged1/N) and3 thenucleotides structure of allowed the 3′- 1 usneighboring to estimate nucleotide the influence (Exo-aPu of the2-X/N opposite-strand) on the 3′-5′ nucleotideexonuclease (Exo-aPu reaction./N ) and the structure of the 30-neighboring nucleotide (Exo-aPu2-X/N) on the 30-50 exonuclease reaction. 2.1. The Position of aPu in the Duplex Affects APE1 3′-Exonuclease Efficiency 2.1. The Position of aPu in the Duplex Affects APE1 30-Exonuclease Efficiency To determine the effect of a position of the aPu nucleotide in the duplex on the rate of its APE1- To determine the effect of a position of the aPu nucleotide in the duplex on the rate of its catalyzed removal, fluorescence kinetic traces were obtained for each of the duplexes Exo-aPuj/T at APE1-catalyzed removal, fluorescence kinetic traces were obtained for each of the duplexes Exo-aPuj/T different concentrations of APE1 using the stopped-flow instrument (Figure 3). For all substrates, at different concentrations of APE1 using the stopped-flow instrument (Figure3). For all substrates, Exo-aPuj/T, the aPu fluorescence intensity increased with time, but the rate of this process decreased Exo-aPuj/T, the aPu fluorescence intensity increased with time, but the rate of this process decreased with the increase of the j value, i.e., with the moving of aPu away from the 3′ end of the short with the increase of the j value, i.e., with the moving of aPu away from the 30 end of the short oligonucleotide. This result can be attributed to the process of sequential removal of non-fluorescent oligonucleotide. This result can be attributed to the process of sequential removal of non-fluorescent nucleotides beginning at the 3′ end until APE1 reaches the aPu nucleotide inside the sequence. To nucleotides beginning at the 30 end until APE1 reaches the aPu nucleotide inside the sequence. support this conclusion and find a possible correlation of the changes of aPu fluorescence intensity with the process of detachment of 3′ nucleotides, hydrolytic cleavage of 5′-32P-labeled 15 nt oligonucleotide of substrate Exo-aPu1/T was studied by polyacrylamide gel electrophoresis (PAGE, Figure 4). Fast accumulation of the total reaction products at various concentrations of substrate Exo- aPu1/T (Figure 4a) coincides with the time point of the aPu fluorescence intensity increase detected for this substrate. Moreover, as shown in Figure 4b,c, PAGE analysis allowed us to resolve the appearance and disappearance of shortened intermediate fragments of 5′-32P-labeled 15 nt

Molecules 2018, 23, 2101 4 of 14

To support this conclusion and find a possible correlation of the changes of aPu fluorescence intensity with the process of detachment of 30 nucleotides, hydrolytic cleavage of 50-32P-labeled 15 nt oligonucleotide of substrate Exo-aPu1/T was studied by polyacrylamide gel electrophoresis (PAGE, Figure4). Fast accumulation of the total reaction products at various concentrations of substrate Exo-aPu1/T (Figure4a) coincides with the time point of the aPu fluorescence intensity Molecules 2018, 23, x 4 of 14 increase detected for this substrate. Moreover, as shown in Figure4b,c, PAGE analysis allowed us to 0 32 oligonucleotideresolve the appearance of substrate and disappearance Exo-aPu1/T ofindicated shortened as intermediateproducts Pn, fragments where n ofis 5the- P-labeledlength of 15 the nt 1 fragmentoligonucleotide in nucleotides. of substrate ComparisonExo-aPu /T ofindicated kinetic da asta products obtained P nby, where fluorescencen is the length(Figure of 3) the and fragment PAGE (Figurein nucleotides. 4) analyses Comparison for substrates of kinetic Exo-aPu data obtainedj/T revealed by fluorescence that the phase (Figure of3) andthe PAGEincrease (Figure in aPu4) j fluorescenceanalyses for substratesintensity coincidedExo-aPu /T withrevealed the removal that the of phase the aPu of the nucleotide increase infrom aPu the fluorescence jth position intensity of the 15coincided nt oligonucleotide. with the removal of the aPu nucleotide from the jth position of the 15 nt oligonucleotide.

(a) (b)

(c) (d)

Figure 3. ChangesChanges in in aPu aPu fluorescence fluorescence intensity intensity during during the the interaction interaction of of APE1 APE1 with one of the 1 2 4 6 substrates: ( (aa)) Exo-aPu1/T/T,,( (b)) Exo-aPu2/T/T,,( (cc)) Exo-aPu4/T/T,, or or ( d) Exo-aPu6/T/T.. Concentrations of of the enzyme and DNA are indicated in the panels.panels. Kineti Kineticc traces for different APE1 concentrations are shown in different colors.

The kinetic parameters of the 3′-5′ exonuclease reaction—resulting in the removal of the 3′ aPu The kinetic parameters of the 30-50 exonuclease reaction—resulting in the removal of the 30 nucleotide from substrates Exo-aPuj/T—were calculated using Equation (3). Kinetic traces in Figure aPu nucleotide from substrates Exo-aPuj/T—were calculated using Equation (3). Kinetic traces in 2a characterizing the interaction of APE1 with substrate Exo-aPu1/T were best fitted by a two- Figure2a characterizing the interaction of APE1 with substrate Exo-aPu1/T were best fitted by a exponent function (Equation (3), i = 2). The observed rate constant k11 of the fast increase phase of aPu two-exponent function (Equation (3), i = 2). The observed rate constant k 1 of the fast increase fluorescence intensity depends on the concentration of APE1 in a hyperbolic1 manner (Figure 5а). phase of aPu fluorescence intensity depends on the concentration of APE1 in a hyperbolic manner Hyperbolic dependence of k11 indicates that the first phase includes a two-step kinetic mechanism. It (Figure5a). Hyperbolic dependence of k 1 indicates that the first phase includes a two-step kinetic is reasonable to suggest that the first step1 reflects a process of initial complex formation between the mechanism. It is reasonable to suggest that the first step reflects a process of initial complex formation enzyme and substrate. Its conversion to catalytically active conformation includes the placement of between the enzyme and substrate. Its conversion to catalytically active conformation includes the a 3′ nucleotide in the enzyme’s active site. The second step in this mechanism could characterize the catalytic reaction of hydrolysis of the phosphodiester bond with the 3′-aPu nucleotide. Using Equation (1), the equilibrium constant of the binding of APE1 to substrate Exo-aPu1/T (Kbind = (1.4 ± 0.2) × 106 М−1) as well as the rate constant of the catalytic reaction (kdetachaPu = 0.096 ± 0.004 s−1) were calculated.

k11 = Kbind × [APE1] × kdetachaPu/(Kbind × [APE1] + 1) (1) Since the 3′-5′ exonuclease reaction proceeds with multiple turnover up to full substrate hydrolysis, it was likely that, after detachment of the aPu nucleotide from the 3′ end of the DNA chain

Molecules 2018, 23, 2101 5 of 14 placement of a 30 nucleotide in the enzyme’s active site. The second step in this mechanism could characterize the catalytic reaction of hydrolysis of the phosphodiester bond with the 30-aPu nucleotide. Using Equation (1), the equilibrium constant of the binding of APE1 to substrate Exo-aPu1/T 6 −1 aPu (Kbind = (1.4 ± 0.2) × 10 M ) as well as the rate constant of the catalytic reaction (kdetach = 0.096 ± 0.004 s−1) were calculated.

1 aPu k1 = Kbind × [APE1] × kdetach /(Kbind × [APE1] + 1) (1)

Molecules 2018, 23, x 5 of 14 Since the 30-50 exonuclease reaction proceeds with multiple turnover up to full substrate hydrolysis, it was likely that, after detachment of the aPu nucleotide from the 30 end of the DNA chain by hydrolytic by hydrolytic cleavage of the 3′-5′ phosphodiester bond, there was a step of removal of the detached cleavage of the 30-50 phosphodiester bond, there was a step of removal of the detached nucleotides from nucleotides from the active site of the enzyme. The rate of the release of a detached aPu nucleotide the active site of the enzyme. The rate of the release of a detached aPu nucleotide could be associated could be associated with the network of contacts of this nucleotide with amino acid residues of the with the network of contacts of this nucleotide with amino acid residues of the active site and most likely active site and most likely should not depend on the concentration of APE1. Calculated values1 of should not depend on the concentration of APE1. Calculated values of observed rate constants k2 do observed rate constants k21 do not reveal their significant dependence on APE1 concentration (Figure not reveal their significant dependence on APE1 concentration (Figure5b). Therefore, we hypothesized 5b). Therefore, we hypothesized that the second phase of the growth of aPu fluorescence intensity that the second phase of the growth of aPu fluorescence intensity represents the release of the detached represents the release of the detached aPu nucleotide from the active site of the enzyme. The linear aPu nucleotide from the active site of the enzyme. The linear approximation of this data allows us to approximation of this data allows us to calculate the average observed rate constant of aPu release 1 aPu −1 calculate the average observed rate constant of aPu release k2 = krelease = 0.0038 ± 0.0002 s . k21 = kreleaseaPu = 0.0038 ± 0.0002 s−1.

(a) (b)

(c)

Figure 4. Kinetics of the accumulation of DNA products during an interaction with APE1 as detected Figure 4. Kinetics of the accumulation of DNA products during an interaction with APE1 as by PAGE. (a) Accumulation of the total reaction products at various concentrations of substrate Exo- detected by PAGE. (a) Accumulation of the total reaction products at various concentrations of aPu1/T, (b) sequential formation of reaction products, and (c) the accumulation and consumption of substrate Exo-aPu1/T,(b) sequential formation of reaction products, and (c) the accumulation and intermediate truncated reaction products Pn during sequential exonuclease removal of 3′-end consumption of intermediate truncated reaction products Pn during sequential exonuclease removal of 3nucleotides.0-end nucleotides.

MoleculesMolecules 20182018,,23 23,, 2101x 66 ofof 1414 Molecules 2018, 23, x 6 of 14

(a) (b) (a) (b) Figure 5. Dependences of the observed rate constants k11 (а1) and k21 (b)1 on APE1 concentration. Data Figure 5. Dependences of the observed rate constants k1 (a) and k2 (b) on APE1 concentration. Figure 5. Dependences of the observed rate constants k11 (а) and k21 (b) on APE1 concentration. Data Datapresented presented in (a) inand (a )(b and) were (b) fitted were to fitted a hyperbolic to a hyperbolic equation equation (Equation (Equation (1)) or to (1)) a linear or to function, a linear presented in (a) and (b) were fitted to a hyperbolic equation (Equation (1)) or to a linear function, function,respectively. respectively. respectively. Thus, the total kinetic mechanism of removal of 3′-aPu during the 3′-5′ exonuclease process is Thus, the total kinetic mechanism of removal of 30-aPu during the 30-50 exonuclease process describedThus, inthe Scheme total kinetic 2. The mechanism complex E·S of in removal Scheme of 2 3corresponds′-aPu during to the the 3 ′catalytic-5′ exonuclease complex process of APE1 is is described in Scheme2. The complex E ·S in Scheme2 corresponds to the catalytic complex of describedwith a DNA in Scheme substrate. 2. TheThe complex3′-aPu detachment E·S in Scheme rate 2constants corresponds calc ulatedto the catalyticby hyperbolic complex fitting of APE1 were APE1 with a DNA substrate. The 30-aPu detachment rate constants calculated by hyperbolic fitting withapproximately a DNA substrate. 25-fold higherThe 3′ -aPuthan detachmentthe rate constant rate ofconstants the aPu calcnucleotideulated by release hyperbolic from the fitting active were site were approximately 25-fold higher than the rate constant of the aPu nucleotide release from the approximatelyof the enzyme, 25-fold which indicateshigher than strong the rate inhibition constant of ofAPE1 the aPuby the nucleotide first detached release nucleotide. from the active Recently site active site of the enzyme, which indicates strong inhibition of APE1 by the first detached nucleotide. ofpublished the enzyme, pre-steady-state which indicates quench strong-flow inhibition analysis of ofAPE1 product by the accumulation first detached in nucleotide. the course Recently of 3′-5′ Recently published pre-steady-state quench-flow analysis of product accumulation in the course of publishedexonuclease pre-steady-state reaction revealed quench a burst-flow phase analysis with aof subsequent product accumulation steady-state linear in the phase course [23], of which 3′-5′ 30-50 exonuclease reaction revealed a burst phase with a subsequent steady-state linear phase [23], exonucleasesupports the reaction notion revealedthat the arate-limiting burst phase withstep ofa subsequent enzyme turnover steady-state is the linear release phase of [23],a detached which which supports the notion that the rate-limiting step of enzyme turnover is the release of a detached supportsnucleotide the from notion the activethat the site. rate-limiting step of enzyme turnover is the release of a detached nucleotide from the active site.

Scheme 2. The kinetic mechanism of removal of 3′-aPu nucleotide. Е is the enzyme, S is a DNA Schemesubstrate, 2. E·SThe is kinetic a complex mechanism of the enzyme of removal with theof 3 substrate,′-aPu nucleotide. Р is a product Е is the of enzyme,substrate S conversion, is a DNA Scheme 2. The kinetic mechanism of removal of 30-aPu nucleotide. E is the enzyme, S is a DNA substrate,E·P is a complex E·S is a of complex the enzyme of the with enzyme the product, with the K bindsubstrate, denotes Р the is a equilibrium product of constantsubstrate of conversion, binding of substrate, E·S is a complex of the enzyme with the substrate, P is a product of substrate conversion, bind E·PAPE1 is a to complex the substrate, of the enzyme kdetachaPu withis a ratethe product,constant Kof the denotes hydrolysis the equilibrium of the 3′-5′ constantphosphodiester of binding bond, of E·P is a complex of the enzyme with the product, Kbind denotes the equilibrium constant of binding of detachaPu APE1and k releaseto theaPu substrate,represents k the rateaPu is constant a rate constant of the release of the ofhydrolysis a detached of theaPu 3 nucleotide′0-5′0 phosphodiester from the bond,active APE1 to the substrate, kdetach is a rate constant of the hydrolysis of the 3 -5 phosphodiester bond, and kreleaseaPu represents the rate constant of the release of a detached aPu nucleotide from the active andsite kof the enzyme.aPu represents the rate constant of the release of a detached aPu nucleotide from the active site ofrelease the enzyme. site of the enzyme. 2.2. Determination of the Mean Rate of Removal of a 3′-End Nucleotide 2.2. Determination of the Mean Rate of Removal of a 30′-End Nucleotide 2.2. DeterminationIn the course ofof the interaction Mean Rate ofof АРЕ Removal1 with of substrates a 3 -End Nucleotide Exo-aPuj/T (Figure 6a), initially the complex In the course of interaction of АРЕ1 with substrates Exo-aPuj/T (Figure 6a), initially the complex of theIn enzyme the course with of interactionthe 3′-end ofnucleotide APE1 with of substrates the substrateExo-aPu is formed.j/T (Figure Then6a), the initially detachment the complex and a of the enzyme with the 3′-end nucleotide of the substrate is formed. Then the detachment and a ofsubsequent the enzyme release with of the the 3 03-end′-end nucleotide nucleotide of takes the substrateplace and, is afte formed.rward, Then the enzyme the detachment is shifted andalong a subsequent release of the 3′-end nucleotide takes place and, afterward, the enzyme is shifted along subsequentthe shortened release DNA of thefragment 30-end to nucleotide the second takes nucleotide place and, and afterward, so far the the enzymej-th aPu isresidue shifted would along the be the shortened DNA fragment to the second nucleotide and so far the j-th aPu residue would be shorteneddetached. DNAIt could fragment be proposed to the secondthat the nucleotide binding of and the so enzyme far the jto-th a aPu substrate—before residue would bethe detached. catalytic detached. It could be proposed that the binding of the enzyme to a substrate—before the catalytic Itaction could and be proposedenzyme dislocation that the binding along DNA of the after enzyme catalysis—proceed to a substrate—before faster than the the catalytic catalytic action reaction. and action and enzyme dislocation along2 DNA after4 catalysis—proceed6 faster than the catalytic reaction. enzymeTherefore, dislocation for substrates along Exo-aPu DNA after/T catalysis—proceed, Exo-aPu /T, and Exo-aPu faster than/T the, the catalytic time required reaction. for Therefore, removal of for a Therefore, for substrates Exo-aPu2/T, Exo-aPu4/T, and Exo-aPu6/T, the time required for removal of a substratesaPu nucleotideExo-aPu placed2/T, Exo-aPu in the middle4/T, and ofExo-aPu the sequen6/T, thece is time equal required to the for sum removal of the of catalytic a aPu nucleotide reaction aPu nucleotide placed in the middle of the sequence is equal to the sum of the catalytic reaction placedperiods in required the middle for ofthe the detachment sequence isof equal the 3 to′-end the sumnucleotides of the catalytic and duration reaction ofperiods the release required of a periods required for the detachment of the 3′-end nucleotides and duration of the release of a fordetached the detachment nucleotide of from the the 30-end active nucleotides site. Due andto th duratione disorder of of the the release enzymatic of a detachedprocess in nucleotidethe course detachedof this repetitive nucleotide multiple from turnoverthe active process site. Due of theto th enzyme,e disorder the ofkineti thec enzymatic curves recorded process for in Exo-aPu the course2/T, from the active site. Due to the disorder of the enzymatic process in the course of this repetitive2 of this repetitive4 multiple turnover6 process of the enzyme, the kinetic curves recorded for Exo-aPu /T, multipleExo-aPu turnover/T, and Exo-aPu process/T of were the enzyme, described the with kinetic a multi-exponential curves recorded function for Exo-aPu (Equation2/T, Exo-aPu (3), i >4 /T3)., Exo-aPu4/T, and Exo-aPu6/T were described with a multi-exponential function (Equation (3), i > 3). andTherefore,Exo-aPu we6/T analyzedwere described only the withfirst exponential a multi-exponential term in this function equation, (Equation which (3), containedi > 3). Therefore, the observed we Therefore,rate constant we analyzedk1j. It is worth only the noting first exponentialthat the values term of in rate this constantsequation, whichk11 and contained k12 (characterizing the observed the rate constant k1j. It is worth noting that the values of rate constants k11 and k12 (characterizing the

Molecules 2018, 23, 2101 7 of 14 analyzed only the first exponential term in this equation, which contained the observed rate constant j 1 2 Moleculesk1 . It is 2018 worth, 23, notingx that the values of rate constants k1 and k1 (characterizing the removal of7 of the 14 first and the second nucleotide) differed approximately 35-fold. On the basis of the kinetic law for the removalrate of the of sequential the first and reactions the second [38], thenucleotide) following diff Equationered approximately (2) is true. 35-fold. On the basis of the kinetic law for the rate of the sequential reactions [38], the following Equation (2) is true. j 1/k1 = j/kdetach + (j − 1)/krelease = j × (1/kdetach + 1/krelease) − 1/krelease (2) 1/k1j = j/kdetach + (j − 1)/krelease = j × (1/kdetach + 1/krelease) − 1/krelease (2)

0 jj where jj isis thethe positionposition ofof aPuaPu fromfrom the the 3 3′end, end,k k11 isis thethe observedobserved raterate constantconstant of of the the first first phase, phase,k detachkdetach is the mean rate constant of the catalyticcatalytic reaction of hydrolysis of a 330′-5′0 phosphodiester bond of any 0 nucleotide at the 3′ end ofof thethe shortshort oligonucleotideoligonucleotide inin DNADNA substrate,substrate, andand kkreleaserelease isis the the mean rate constant of a release of any detached nucleotide from thethe activeactive sitesite ofof APE1.APE1. j Based on Figure 66b,b, thethe reversereverse valuevalue ofof kk11j linearly depends depends on on j,, which allows us to calculate −1 −1 −1 −1 the values values of of kdetachkdetach = 0.08= 0.08 ± 0.02± 0.02s and s kreleaseand =k 0.0035release = ± 0.0001 0.0035 s± using0.0001 Equation s using (2). Equation Thus, these (2). results Thus, revealthese results that the reveal hydrolysis that the of hydrolysis a phosphodiester of a phosphodiester bond of any 3 bond′-end of nucleotide any 30-end proceeds nucleotide at least proceeds 10-fold at fasterleast 10-fold than the faster release than of the this release detached of this nucleotide detached from nucleotide the active from site the of activeAPE1. site The of rate APE1. constants The rate of detachmentconstants of and detachment release of and any releasenucleotide of any are nucleotidein good agreement are in good with agreementthe constants with obtained the constants for the 1 aPuobtained nucleotide for the by aPu analysis nucleotide involving by analysis a set of involving concentrations a set of of concentrations substrate Exo-aPu of substrate1/T (kdetachExo-aPuaPu = 0.096/T aPu−1 aPu −1 −aPu1 −1 (±k 0.004detach s and= 0.096 krelease± 0.004 = 0.0038 s and ± 0.0002krelease s ). = 0.0038 ± 0.0002 s ).

(a) (b)

Figure 6. TheThe change change in in the the fluorescence fluorescence intensity of aPu in the course of APE1 interaction with j substrates Exo-aPuExo-aPuj/T.(. (aa)) ExponentialExponential analysisanalysis (red (red line) line) of of the the initial initial part part of of the the kinetic kinetic curves curves (j is ( thej is j positionthe position of the of nucleotidethe nucleotide relative relative to the to 3the0 end 3′ ofend the ofshort the short oligonucleotide). oligonucleotide). [Exo-aPu [Exo-aPuj/T] =/T 1.0] =µ 1.0M, μM, [APE1] = 2.0 μM. (b) Dependence jof k1j on j, which is described by Equation (2). [APE1] = 2.0 µM. (b) Dependence of k1 on j, which is described by Equation (2).

2.3. The Rate of the 3′-5′ Exonuclease Reaction Depends on Stability of the 3′-End Base Pair 2.3. The Rate of the 30-50 Exonuclease Reaction Depends on Stability of the 30-End Base Pair It has been shown earlier [32,39] that the efficiency of a 3′-5′ exonuclease reaction depends on It has been shown earlier [32,39] that the efficiency of a 30-50 exonuclease reaction depends on the the thermal stability of DNA duplexes. In this paper, we studied the effect of the terminal 3′-base pair thermal stability of DNA duplexes. In this paper, we studied the effect of the terminal 30-base pair on the removal of the first 3′-end aPu nucleotide. For this purpose, duplexes Exo-aPu1/N containing on the removal of the first 30-end aPu nucleotide. For this purpose, duplexes Exo-aPu1/N containing bases cytosine, thymine, adenine, and guanine opposite aPu (N = C, Т, A, and G) were used. The bases cytosine, thymine, adenine, and guanine opposite aPu (N = C, T, A, and G) were used. The kinetic traces (Figure 7a) obtained for the interactions of APE1 (1.0 μM) with duplexes Exo-aPu1/N kinetic traces (Figure7a) obtained for the interactions of APE1 (1.0 µM) with duplexes Exo-aPu1/N (1.0 μM) revealed that the rate of aPu removal depends on the opposite base. (1.0 µM) revealed that the rate of aPu removal depends on the opposite base. The kinetic traces presented in Figure 7a were estimated using Equation (3) where i = 2 and the The kinetic traces presented in Figure7a were estimated using Equation (3) where i = 2 and values of the observed rate constants of the first and the second phases k11 and k21, respectively, were the values of the observed rate constants of the first and the second phases k 1 and k 1, respectively, 1 1 2 calculated. Following from the preceding discussion, the rate constant k1 characterizes1 DNA binding were calculated. Following from the preceding discussion, the rate constant k1 characterizes DNA and detachment of the 3′-aPu nucleotide while k21 reflects the release of an aPu nucleotide. As binding and detachment of the 30-aPu nucleotide while k 1 reflects the release of an aPu nucleotide. 2 1 presented in Table 1 and Figure 7b, only the rate constant of 3′-end0 aPu detachment k1 depends1 on As presented in Table1 and Figure7b, only the rate constant of 3 -end aPu detachment k1 depends the opposite base and increases in the order of T < C < A < G. It was shown in Reference [40] that the on the opposite base and increases in the order of T < C < A < G. It was shown in Reference [40] stability of an aPu pair with one of the natural DNA bases decreases in the same order. In fact, 2- aminopurine forms two hydrogen bonds with thymine or one hydrogen bond with cytosine, but it does not form hydrogen bonds with purine bases. Therefore, our data are consistent with the conclusion that the formation of a catalytically active complex of APE1 proceeds faster with the substrates containing a mismatched purine base opposite of 2-aminopurine, i.e., aPu/G and aPu/А

Molecules 2018, 23, 2101 8 of 14 that the stability of an aPu pair with one of the natural DNA bases decreases in the same order. In fact, 2-aminopurine forms two hydrogen bonds with thymine or one hydrogen bond with cytosine, butMolecules it does 2018, not23, x form hydrogen bonds with purine bases. Therefore, our data are consistent8 with of 14 the conclusion that the formation of a catalytically active complex of APE1 proceeds faster with the substratesdue to the absence containing of any a mismatched complementary purine interactions base opposite and ofdestabilization 2-aminopurine, of the i.e., 3 aPu/G′ end. The and obtained aPu/A duedata toare the in absence agreement of any with complementary recent structural interactions data [23] and destabilizationsuggesting that of APE1 the 30 end.likely The removes obtained a datamatching are in base agreement through with the recentsame mechanism structural data as a [ 23mismatched] suggesting base that only APE1 at likelylower removes efficiency a matchingowing to basethe relative through lack the of same flexibility mechanism at the as3′ aend. mismatched It was shown base in only this at study lower that efficiency the stable owing C/G to base the relativepairing lackprevents of flexibility the phosphate at the 3backbone0 end. It was from shown entering in this the studyproper that registry the stable for cleavage C/G base by pairingthe APE1 prevents active thesite. phosphateA comparison backbone of structures from entering of the APE1 the proper complex registry with formatching cleavage C/G by and the APE1mismatched active C/T site. Asubstrates comparison reveals of structures that the matched of the APE1 C is complex shifted with7.5 Å matchingdownstream C/G and and away mismatched from the C/T key substrates catalytic revealsresidues. that the matched C is shifted 7.5 Å downstream and away from the key catalytic residues. 1 The observed rate constants of the secondsecond phasephase kk221 expectedlyexpectedly do do not depend on the nature of the oppositeopposite nucleotidenucleotide because because this this phase phase reflects reflects the the aPu aPu nucleotide nucleotide release release from from the the active active site site of the of 1 1 −1 enzyme.the enzyme. The The calculated calculated value value of kof2 kfor21 for all all substrates substratesExo-aPu Exo-aPu/N1/Nwas was equal equal to to0.0034 0.0034± ±0.0002 0.0002 s s−1 and was very close to the same value determined for an aPu release by analysis involving a set of 1 aPu −1 concentrations ofof substratesubstrateExo-aPu Exo-aPu/T1/T( k(releasekreleaseaPu == 0.0038 0.0038 ±± 0.00020.0002 s s−1, Figure, Figure 5b)5b) and and the mean raterate 0 −1 constant of a release of any 3′-end nucleotide (krelease == 0.0035 0.0035 ± ±0.00010.0001 s−1 s, Figure, Figure 6b).6b).

(b) (a)

Figure 7. (a) A change in the intensity of aPu in the course of APE1 interaction with substrates Exo- Figure 7. (a) A change in the intensity of aPu in the course of APE1 interaction with substrates 1 1 1 aPu /N. (1b) The values of the observed rate constants ki ([APE1]1 = 1.0 μM, [Exo-aPu /N1] = 1.0 μM). Exo-aPu /N. (b) The values of the observed rate constants ki ([APE1] = 1.0 µM, [Exo-aPu /N] = 1.0 µM).

1 Table 1.1. The rate constants of the interactioninteraction ofof APE1APE1 withwith substratessubstrates Exo-aPuExo-aPu1/N.

DNA DNA Binding and aPu Detaching, k11, s−1 aPu Releasing, k21, s−1 DNA DNA Binding and aPu Detaching, k 1, s−1 aPu Releasing, k 1, s−1 Exo-aPu1/G 0.13 ± 0.02 1 0.0035 ± 0.00022 1 Exo-aPuExo-aPu 1/G/A 0.0750.13 ± ± 0.020.001 0.0035 0.0035± ±0.0002 0.0002 Exo-aPu1/A ± ± Exo-aPu1/C 0.0750.050 ± 0.0010.001 0.0035 0.0031 ±0.0002 0.0002 Exo-aPu1/C 0.050 ± 0.001 0.0031 ± 0.0002 1 Exo-aPuExo-aPu1//TT 0.0400.040± ± 0.0010.001 0.0034 0.0034± ±0.0002 0.0002

2.4. The Rate of the 3 ′0-End Nucleotide Removal DependsDepends onon ItsIts StructureStructure For the assay of the impact of the 3′-end nucleotide structure on the rate of its removal, duplexes For the assay of the impact of the 30-end nucleotide structure on the rate of its removal, duplexes Exo-aPu2-X/N were used as DNA substrates. These duplexes contained an aPu nucleotide at the Exo-aPu2-X/N were used as DNA substrates. These duplexes contained an aPu nucleotide at the second position from the 3′ end, but the first position was occupied by nucleotide X and contained second position from the 30 end, but the first position was occupied by nucleotide X and contained either an undamaged base (adenine (A), guanine (G), cytosine (C), or thymine (T)) or a modified base: either an undamaged base (adenine (A), guanine (G), cytosine (C), or thymine (T)) or a modified base: 8-oxoguanine (oxoG), uracil (U), 5-methylcytosine (5mC), -anomer of adenosine (αA), or 8-oxoguanine (oxoG), uracil (U), 5-methylcytosine (5mC), α-anomer of adenosine (αA), or representing representing the 2-oxymethyl-3-oxy-tetrahydrofurane residue (F) or phosphate (p). the 2-oxymethyl-3-oxy-tetrahydrofurane residue (F) or phosphate (p). The aPu fluorescence kinetic curves obtained for the case of interaction of APE1 with substrates Exo-aPu2-X/N are presented in Figure 8a,b. The initial exponential increase of the aPu fluorescence was fitted to Equation (3) to calculate the observed rate constant k12 (Figure 8c and Table 2). It is evident from these data that k12 changes in a rather narrow range (0.5–4.6) × 10−3 s−1. According to previous analysis, k12 may characterize a combination of processes of DNA binding, detachment and release of the 3′-X nucleotide, and subsequent detachment of the aPu

Molecules 2018, 23, 2101 9 of 14

The aPu fluorescence kinetic curves obtained for the case of interaction of APE1 with substrates Exo-aPu2-X/N are presented in Figure8a,b. The initial exponential increase of the aPu fluorescence 2 was fitted to Equation (3) to calculate the observed rate constant k1 (Figure8c and Table2). It is 2 −3 −1 evident from these data that k1 changes in a rather narrow range (0.5–4.6) × 10 s . 2 MoleculesAccording 2018, 23, to x previous analysis, k1 may characterize a combination of processes of DNA binding,9 of 14 detachment and release of the 30-X nucleotide, and subsequent detachment of the aPu nucleotide. Innucleotide. the approximation In the approximation where the binding where ofthe different bindingExo-aPu of different2-X/N Exo-aPusubstrates2-X/N can substrates be considered can be the 2 2 same,considered the difference the same, in the the difference rate constant in thek 1rateis constant due to the k1 is difference due to the in difference the rates in of the the rates X nucleotide of the detachmentX nucleotide and detachment release from and the release active site.from Moreover, the active because site. Moreover, the nucleotide because detachment the nucleotide rate is at leastdetachment 20-fold higher rate is thanat least the 20-fold rate of higher the nucleotide than the ra release,te of the it nucleotide could be concluded release, it could that the be releaseconcluded of the that the release of the nucleotide from the active site determines the differences in the2 observed nucleotide from the active site determines the differences in the observed constants k1 . 2 constantsThe rates k1 . of removal of the aPu nucleotide are higher when it is located after the G/C andThe rates C/G of pairs removal in comparisonof the aPu nucleotide with the are A/T higher and when T/A it is pairs. located Inafter the the case G/C and when C/G aPu pairs in comparison with the A/T and T/A pairs. In the case when2 aPu is located after a damaged X/N is located after a damaged X/N pair, the values of k1 decrease in the following order: pair, the values of k12 decrease in the following order: F/G ≥ αA/T > oxoG/C = U/G > meC/G ≥ p/C, F/G ≥ αA/T > oxoG/C = U/G > meC/G ≥ p/C, which indicates the maximal release rate for the which indicates the maximal release rate for the abasic nucleotide. For duplexes containing base pairs2 abasic nucleotide. For duplexes containing base pairs oxoG/C, U/G, or C/G, the values of k1 are oxoG/C, U/G, or C/G, the values of k12 are very close and are in the range (1.7–2.1) × 10−3 s−1. Otherwise, very close and are in the range (1.7–2.1) × 10−3 s−1. Otherwise, for the duplex with a G/C base pair, for the duplex with a G/C base pair, the observed rate constant k12 is sufficiently higher and equal to the observed rate constant k 2 is sufficiently higher and equal to 2.9 × 10−3 s−1. The minimal value of 2.9 × 10−3 s−1. The minimal value1 of k12 (5.3 × 10−4 s−1) was detected for the removal of a 3′-phosphate 2 −4 −1 0 2 k1group(5.3 × in10 the cases )of was Exo-aPu detected2-p/C for, which the removal indicates of that a 3 the-phosphate 3′-phosphatase group activity in the case of APE1 of Exo-aPu is weaker-p/C , 0 0 0 whichthan indicatesits 3′-5′ exonuclease that the 3 activity.-phosphatase activity of APE1 is weaker than its 3 -5 exonuclease activity.

(a) (b)

(c)

Figure 8. A change in the intensity of aPu in the course of APE1 interaction with substrates Exo-aPu2- Figure 8. A change in the intensity of aPu in the course of APE1 interaction with substrates X/N that2 contain an undamaged (a) or damaged (b) 3′-end nucleotide.0 (c) The values of the observed Exo-aPu -X/N that2 contain an undamaged (a) or damaged (b) 3 -end nucleotide.1 (c) The values of rate constants ki , X/N-pair is2 depicted in figure. [APE1] = 1.0 μM, [Exo-aPu /N] = 1.0 μM. 1 the observed rate constants ki , X/N-pair is depicted in figure. [APE1] = 1.0 µM, [Exo-aPu /N] = 1.0 µM.

Molecules 2018, 23, 2101 10 of 14

2 2 Table 2. The rate constants k1 of the interaction of APE1 with substrates Exo-aPu -X/N.

2 −1 X/N Nature k1 , s F/G (4.6 ± 0.05) × 10−3 oxoG/C (1.7 ± 0.07) × 10−3 p/C (5.3 ± 0.6) × 10−4 5mC/G (8.9 ± 0.5) × 10−4 U/G (1.9 ± 0.06) × 10−3 αA/T (3.3 ± 0.04) × 10−3 A/T (9.9 ± 0.6) × 10−4 T/A (9.2 ± 0.5) × 10−4 C/G (2.1 ± 0.07) × 10−3 G/C (2.9 ± 0.05) × 10−3

3. Materials and Methods

3.1. Protein Expression and Purification To purify APE1 expressed as a recombinant protein, 1 L of culture (in Luria-Bertani (LB) broth) of the Escherichia coli strain Rosetta II(DE3) (Merck KGaA, Darmstadt, Germany) carrying the pET11a-APE1 construct was grown with 50 µg/mL ampicillin at 37 ◦C until absorbance at 600 nm (A600) reached 0.6 to 0.7. APE1 expression was induced overnight with 0.2 mM isopropyl-β-D-thiogalactopyranoside. The method for isolation of wild-type APE1 has been described previously (for more details, see Supplementary Materials: Figure S1) [41]. The protein concentration was measured by the Bradford method [42]. The stock solution was stored at −20 ◦C.

3.2. Oligodeoxyribonucleotides The sequences of oligodeoxyribonucleotides used in this work are listed in Table3. The oligodeoxyribonucleotides were synthesized by the standard phosphoramidite method on an ASM-700 synthesizer (BIOSSET, Novosibirsk, Russia) from phosphoramidites purchased from Glen Research (Sterling, VA, USA). α-20-Deoxyadenosine phosphoramidite was bought from ChemGenes Corp. (Wilmington, MA, USA). Synthetic oligonucleotides were unloaded from the solid support with ammonium hydroxide, according to manufacturer protocols. Deprotected oligonucleotides were purified by HPLC. Concentrations of oligonucleotides were calculated from their absorbance at 260 nm. Oligodeoxyribonucleotide duplexes were prepared by annealing modified and complementary strands at a 1:1 molar ratio. Shorthands of DNA duplexes denote the number of position for aPu residue from the 30 end in the short oligonucleotide and letter “N”, which correspond to the T, A, G, or C nucleotide placed opposite to the 30 end of the short oligonucleotide in the 50 dangling oligonucleotide.

Table 3. DNA duplexes used as substrates and ligands of APE1.

Shorthand Sequence Exo-aPu1/N 50-CAGCTCTGTACGTG(aPu)-30 N = A, G, C, T 30-GTCGAGACATGCAC N CGTCACCACTGTG-50 50-CAGCTCTGTACGT(aPu)A-30 Exo-aPu2/T 30-GTCGAGACATGCA C TCGTCACCACTGTG-50 50-CAGCTCTGTAC(aPu)TGA-30 Exo-aPu4/T 30-GTCGAGACATG C ACTCGTCACCACTGTG-50 50-CAGCTCTGT(aPu)CGTGA-30 Exo-aPu6/T 30-GTCGAGACA T GCACTCGTCACCACTGTG-50 Exo-aPu2-X/N 50-CAGCTCTGTACGT(aPu)X-30 N = A, G, C, T 30-GTCGAGACATGCA C NCGTCACCACTGTG-50 X = F, G, C, T, oxoG, U, 5mC, p, αA Molecules 2018, 23, 2101 11 of 14

3.3. Stopped-Flow Experiments These experiments were conducted essentially as described previously [43–45]. An SX.18MV stopped-flow spectrometer (Applied Photophysics, Leatherhead, UK) fitted with a 150 W Xe arc lamp and a 2 mm path length optical cell was employed. The dead time of the instrument was 1.4 ms. The excitation wavelength was 310 nm for the aPu fluorescent dye. The emission was monitored using a long-pass wavelength filter (Corion, Franklin, MA, USA) at 370 nm. APE1 was placed in one of the instrument0s syringes and rapidly mixed with the substrate in another syringe. The reported concentrations of reactants are those in the reaction chamber after mixing. Typically, each trace shown in the figures is the average of four or more fluorescence traces recorded in individual experiments. In the figures, if necessary for better presentation, the curves were manually moved apart. All the experiments were carried out in a buffer consisting of 50 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM ◦ MgCl2, 1 mM EDTA, 1 mM DTT, and 7% glycerol (v/v) at 37 C.

3.4. Product Analysis To analyze the products formed by APE1, the substrates were 50-32P-labeled with phage T4 polynucleotide kinase and γ-32P-ATP. The reaction was carried out under the conditions described above. Cleavage of the substrate was initiated by the addition of APE1. Aliquots (2 µL) of the reaction mixture were taken at certain time intervals, immediately quenched with 3 µL of a gel-loading dye containing 7 M urea and 50 mM EDTA, and loaded on a 20% (w/v) polyacrylamide gel containing 7 M urea. Disappearance of the substrate and formation of the products were analyzed by autoradiography and quantified by scanning densitometry in the Gel-Pro Analyzer software, v.4.0 (Media Cybernetics, Rockville, MD, USA).

3.5. Kinetic Data Analysis Stopped-flow kinetic traces were fitted to Equation (3) by a nonlinear regression procedure in the Origin software (OriginLab Corp., Northampton, MA, USA).

N Fc = Fb + ∑ Ai × exp (−ki × t) (3) i=0 where Fc is the observed fluorescence intensity of aPu, Fb indicates background fluorescence, Ai denotes fluorescence parameters, ki is the observed rate constant, and t is the reaction time.

4. Conclusions In general, our findings revealed that in a 30-50 exonuclease reaction, removal of the first 30-terminal nucleotide proceeds significantly faster (approximately 35-fold) than the removal of subsequent nucleotides located at the next positions from the 30 end. This finding indicates that catalysis during the first enzymatic turnover is more rapid than in the subsequent enzymatic cycles, which supports the appearance of the rate-limiting step after catalytic hydrolysis of the phosphodiester bond. It is reasonable to conclude that the deceleration of the removal rate for the subsequent nucleotides in comparison with the first one is caused by a slow release of the detached nucleotide from the active site of the enzyme. It was shown that the rate of aPu removal from the 30 end depends inversely on the stability of the aPu pair with undamaged DNA bases. The obtained kinetic data show that the effect of pair stability is relatively moderate and a difference in the observed rate constant does not exceed five-fold, which suggests that formation of the catalytic complex with any pairs at the 30 end proceeds rapidly and efficiently. A comparison of various undamaged and damaged 30-end nucleotides revealed that the rate of the release of the detached nucleotide from the active site of the enzyme varies ≤10-fold with maximum and minimum values corresponding to an abasic nucleotide and phosphate group, respectively. Most likely this variation is associated with differences in the nucleotide structure. Taken together, our results are suggestive of a kinetic mechanism of the 30-50 Molecules 2018, 23, 2101 12 of 14 exonuclease reaction and indicate that the release of a detached nucleotide from the active site of APE1 is the rate-limiting step of APE1 30-50 exonuclease activity.

Supplementary Materials: The following are available online: Figure S1. Author Contributions: N.A.K. conceived and designed the experiments. A.A.K. conducted the experiments. A.A.K., N.A.K., and O.S.F. analyzed the data. N.A.K. and O.S.F. contributed reagents, materials, and/or analytical tools and A.A.K., N.A.K. and O.S.F. wrote the paper. Conceptualization, N.A.K., and O.S.F. Methodology, A.A.K. Formal Analysis, A.A.K. and N.A.K. Resources, O.S.F. Writing—Original Draft Preparation, A.A.K., N.A.K., and O.S.F. Funding: This work was supported partially by a Russian-Government-funded project (No. VI.57.1.2, 0309-2016-0001) and Russian Foundation of Basic Research grant No. 16-04-00037. The part of the work with aPu detection combined with stopped-flow kinetics was specifically funded by the Russian Science Foundation grant No. 18-14-00135. Conflicts of Interest: The authors declare no conflict of interest.

References

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Sample Availability: Samples of the compounds are not available from the authors.

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