TY - JOUR

T1 - Apurinic/apyrimidinic endonuclease Apn1 from Saccharomyces cerevisiae is recruited to the nucleotide incision repair pathway

T2 - Kinetic and structural features

AU - Dyakonova, Elena S.

AU - Koval, Vladimir V.

AU - Lomzov, Alexander A.

AU - Ishchenko, Alexander A.

AU - Fedorova, Olga S.

N1 - Publisher Copyright: © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)

PY - 2018/9/1

Y1 - 2018/9/1

N2 - Apurinic/apyrimidinic endonuclease Apn1 of Saccharomyces cerevisiae is known as a key player of the base excision DNA repair (BER) pathway in yeast. BER is initiated by DNA glycosylases, whereas Apn1 can start DNA repair individually in the nucleotide incision repair (NIR) pathway. The aim of this research was to elucidate kinetic and structural dynamic aspects of Apn1 involvement in the NIR process. One of the key characteristics of AP endonuclease's interactions is known to be divalent metal ions playing a part of a cofactor. Well-studied human APE1 employs Mg2+ ions, with metal ion concentration's affecting enzymatic activity exerted by APE1. In our study, we aimed to test the effect of the Mg2+ ion on Apn1's NIR catalysis by examining structural dynamics of DNA during the interaction in real time using the stopped-flow technique. To test NIR activity of Apn1, deoxyribooligonucleotide duplexes containing a 5,6-dihydro-2′-deoxyuridine (DHU) residue were employed as substrates. A 2-aminopurine (2-aPu) residue was a reporter group fluorescence intensity of which was detected during Apn1–DNA interactions. NIR activity of both WT and H83A Apn1 was found to be arrested during the interaction with a DNA duplex containing the 2-aPu residue upstream of DHU. We conducted molecular dynamics simulations to elucidate the structural features of complexes of the enzyme with DHU-containing DNAs. The NIR recruiting S. cerevisiae Apn1 proceeds via multistep rearrangements of the complex of Apn1 with a DHU-containing DNA substrate and results in the incised product of the reaction. For wild-type Apn1, the catalytic rate constants do not depend on the Mg2+ concentration, i.e., they are equal in NIR and BER buffers, with equilibrium association constant Ka being 10-fold higher in NIR buffer. Our data reveal more delicate regulation of Apn1's NIR activity due to the more complicated kinetic mechanism, as compared to BER.

AB - Apurinic/apyrimidinic endonuclease Apn1 of Saccharomyces cerevisiae is known as a key player of the base excision DNA repair (BER) pathway in yeast. BER is initiated by DNA glycosylases, whereas Apn1 can start DNA repair individually in the nucleotide incision repair (NIR) pathway. The aim of this research was to elucidate kinetic and structural dynamic aspects of Apn1 involvement in the NIR process. One of the key characteristics of AP endonuclease's interactions is known to be divalent metal ions playing a part of a cofactor. Well-studied human APE1 employs Mg2+ ions, with metal ion concentration's affecting enzymatic activity exerted by APE1. In our study, we aimed to test the effect of the Mg2+ ion on Apn1's NIR catalysis by examining structural dynamics of DNA during the interaction in real time using the stopped-flow technique. To test NIR activity of Apn1, deoxyribooligonucleotide duplexes containing a 5,6-dihydro-2′-deoxyuridine (DHU) residue were employed as substrates. A 2-aminopurine (2-aPu) residue was a reporter group fluorescence intensity of which was detected during Apn1–DNA interactions. NIR activity of both WT and H83A Apn1 was found to be arrested during the interaction with a DNA duplex containing the 2-aPu residue upstream of DHU. We conducted molecular dynamics simulations to elucidate the structural features of complexes of the enzyme with DHU-containing DNAs. The NIR recruiting S. cerevisiae Apn1 proceeds via multistep rearrangements of the complex of Apn1 with a DHU-containing DNA substrate and results in the incised product of the reaction. For wild-type Apn1, the catalytic rate constants do not depend on the Mg2+ concentration, i.e., they are equal in NIR and BER buffers, with equilibrium association constant Ka being 10-fold higher in NIR buffer. Our data reveal more delicate regulation of Apn1's NIR activity due to the more complicated kinetic mechanism, as compared to BER.

KW - AP endonuclease

KW - Base excision repair

KW - DNA repair

KW - Nucleotide incision repair

KW - Saccharomyces cerevisiae

KW - Stopped-flow technique

KW - SUBSTRATE SPECIFICITIES

KW - ESCHERICHIA-COLI

KW - CONFORMATIONAL DYNAMICS

KW - 2-AMINOPURINE

KW - APURINIC ENDONUCLEASE

KW - DNA LESION RECOGNITION

KW - ENZYME

KW - ABASIC SITES

KW - BASE DAMAGE

KW - CATALYTIC MECHANISM

KW - Saccharomyces cerevisiae Proteins/chemistry

KW - Magnesium/metabolism

KW - Catalysis

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry

KW - Molecular Dynamics Simulation

KW - Saccharomyces cerevisiae/enzymology

KW - DNA Repair

KW - Protein Conformation

KW - DNA Damage

KW - Kinetics

UR - http://www.scopus.com/inward/record.url?scp=85049308299&partnerID=8YFLogxK

U2 - 10.1016/j.biochi.2018.06.012

DO - 10.1016/j.biochi.2018.06.012

M3 - Article

C2 - 29959063

AN - SCOPUS:85049308299

VL - 152

SP - 53

EP - 62

JO - Biochimie

JF - Biochimie

SN - 0300-9084

ER -