TY - JOUR

T1 - The mechanism of damage recognition by apurinic/apyrimidinic endonuclease Nfo from Escherichia coli

AU - Senchurova, Svetlana I.

AU - Syryamina, Victoria N.

AU - Kuznetsova, Aleksandra A.

AU - Novopashina, Darya S.

AU - Ishchenko, Alexander A.

AU - Saparbaev, Murat

AU - Dzuba, Sergei A.

AU - Fedorova, Olga S.

AU - Kuznetsov, Nikita A.

N1 - Funding Information: Nikita Kuznetsov reports financial support was provided by Institute of Chemical Biology and Fundamental Medicine Siberian Branch of the Russian Academy of Sciences. Funding Information: This work was supported partially by a Russian-Government –funded project (No. 121031300041-4 ), by Electricité de France ( RB 2020-02 and RB 2021-05 , to M.S), by French National Research Agency (ANR-18-CE44-0008) and Fondation ARC ( PJA-2021060003796 ) to A.A.I. The part of this work involving FRET detection combined with stopped-flow kinetics was specifically funded by Russian Science Foundation grant No. 21-64-00017 . V.N.S. and S.A.D. (Voevodsky Institute of Chemical Kinetics and Combustion SB RAS) acknowledge the core funding from the Russian Federal Ministry of Science and Higher Education (project no. AAAA-A21-121011390038-1 ). Publisher Copyright: © 2022

PY - 2022/11

Y1 - 2022/11

N2 - Apurinic/apyrimidinic (AP) endonuclease Nfo from Escherichia coli recognises AP sites in DNA and catalyses phosphodiester bond cleavage on the 5′ side of AP sites and some damaged or undamaged nucleotides. Here, the mechanism of target nucleotide recognition by Nfo was analysed by pulsed electron–electron double resonance (PELDOR, also known as DEER) spectroscopy and pre–steady-state kinetic analysis with Förster resonance energy transfer detection of DNA conformational changes during DNA binding. The efficiency of endonucleolytic cleavage of a target nucleotide in model DNA substrates was ranked as (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran [F-site] > 5,6-dihydro-2′-deoxyuridine > α-anomer of 2′-deoxyadenosine >2′-deoxyuridine > undamaged DNA. Real-time conformational changes of DNA during interaction with Nfo revealed an increase of distances between duplex ends during the formation of the initial enzyme–substrate complex. The use of rigid-linker spin-labelled DNA duplexes in DEER measurements indicated that double-helix bending and unwinding by the target nucleotide itself is one of the key factors responsible for indiscriminate recognition of a target nucleotide by Nfo. The results for the first time show that AP endonucleases from different structural families utilise a common strategy of damage recognition, which globally may be integrated with the mechanism of searching for specific sites in DNA by other enzymes.

AB - Apurinic/apyrimidinic (AP) endonuclease Nfo from Escherichia coli recognises AP sites in DNA and catalyses phosphodiester bond cleavage on the 5′ side of AP sites and some damaged or undamaged nucleotides. Here, the mechanism of target nucleotide recognition by Nfo was analysed by pulsed electron–electron double resonance (PELDOR, also known as DEER) spectroscopy and pre–steady-state kinetic analysis with Förster resonance energy transfer detection of DNA conformational changes during DNA binding. The efficiency of endonucleolytic cleavage of a target nucleotide in model DNA substrates was ranked as (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran [F-site] > 5,6-dihydro-2′-deoxyuridine > α-anomer of 2′-deoxyadenosine >2′-deoxyuridine > undamaged DNA. Real-time conformational changes of DNA during interaction with Nfo revealed an increase of distances between duplex ends during the formation of the initial enzyme–substrate complex. The use of rigid-linker spin-labelled DNA duplexes in DEER measurements indicated that double-helix bending and unwinding by the target nucleotide itself is one of the key factors responsible for indiscriminate recognition of a target nucleotide by Nfo. The results for the first time show that AP endonucleases from different structural families utilise a common strategy of damage recognition, which globally may be integrated with the mechanism of searching for specific sites in DNA by other enzymes.

KW - 5,6-dihydro-2′-deoxyuridine

KW - Abasic site

KW - Apurinic/apyrimidinic endonuclease

KW - Conformational dynamics

KW - Damaged DNA

KW - DEER spectroscopy

KW - DNA repair

KW - FRET

KW - Stopped-flow enzyme kinetics

KW - α-2′-deoxyadenosine

KW - Electron Spin Resonance Spectroscopy

KW - Deoxyuridine

KW - Escherichia coli

KW - Humans

KW - Nucleotides

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase

KW - DNA

KW - DNA Repair

KW - Endonucleases

KW - DNA Damage

KW - Kinetics

UR - http://www.scopus.com/inward/record.url?scp=85136339002&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/f21cb43d-e230-34ab-999e-16c13dd040e6/

U2 - 10.1016/j.bbagen.2022.130216

DO - 10.1016/j.bbagen.2022.130216

M3 - Article

C2 - 35905924

AN - SCOPUS:85136339002

VL - 1866

JO - Biochimica et Biophysica Acta - General Subjects

JF - Biochimica et Biophysica Acta - General Subjects

SN - 0304-4165

IS - 11

M1 - 130216

ER -