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5' Exonuclease Activity of Human AP

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5' Exonuclease Activity of Human AP molecules Article Kinetic Features of 30-50 Exonuclease Activity of Human AP-Endonuclease 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 enzymes 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 nucleotide 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 enzyme’s active site. 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 nucleotides 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 amino acid 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.
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