TY - JOUR

T1 - Inner Amino Acid Contacts Are Key Factors of Multistage Structural Rearrangements of DNA and Affect Substrate Specificity of Apurinic/Apyrimidinic Endonuclease APE1

AU - Bulygin, Anatoly A

AU - Syryamina, Victoria N

AU - Kuznetsova, Aleksandra A

AU - Novopashina, Darya S

AU - Dzuba, Sergei A

AU - Kuznetsov, Nikita A

N1 - Funding: V.N.S. and S.A.D. (Voevodsky Institute of Chemical Kinetics and Combustion SB RAS) acknowledge the core funding from the Russian Ministry of Science and Higher Education (FWGF-FWGF-2021-0003). This work was partially supported by Russian Ministry of Science and Higher Education project No. 121031300041-4 (for N.A.K.). The part of the work involving the kinetic analysis of the zAPE1 variants was specifically funded by Russian Science Foundation grant No. 23-44-00064.

PY - 2023/7/14

Y1 - 2023/7/14

N2 - Apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most important enzymes in base excision repair. Studies on this enzyme have been conducted for a long time, but some aspects of its activity remain poorly understood. One such question concerns the mechanism of damaged-nucleotide recognition by the enzyme, and the answer could shed light on substrate specificity control in all enzymes of this class. In the present study, by pulsed electron-electron double resonance (DEER, also known as PELDOR) spectroscopy and pre-steady-state kinetic analysis along with wild-type (WT) APE1 from Danio rerio (zAPE1) or three mutants (carrying substitution N253G, A254G, or E260A), we aimed to elucidate the molecular events in the process of damage recognition. The data revealed that the zAPE1 mutant E260A has much higher activity toward DNA substrates containing 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), or 1,N6-ethenoadenosine (εA). Examination of conformational changes in DNA clearly revealed multistep DNA rearrangements during the formation of the catalytic complex. These structural rearrangements of DNA are directly associated with the capacity of damaged DNA for enzyme-induced bending and unwinding, which are required for eversion of the damaged nucleotide from the DNA duplex and for its placement into the active site of the enzyme. Taken together, the results experimentally prove the factors that control substrate specificity of the AP endonuclease zAPE1.

AB - Apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most important enzymes in base excision repair. Studies on this enzyme have been conducted for a long time, but some aspects of its activity remain poorly understood. One such question concerns the mechanism of damaged-nucleotide recognition by the enzyme, and the answer could shed light on substrate specificity control in all enzymes of this class. In the present study, by pulsed electron-electron double resonance (DEER, also known as PELDOR) spectroscopy and pre-steady-state kinetic analysis along with wild-type (WT) APE1 from Danio rerio (zAPE1) or three mutants (carrying substitution N253G, A254G, or E260A), we aimed to elucidate the molecular events in the process of damage recognition. The data revealed that the zAPE1 mutant E260A has much higher activity toward DNA substrates containing 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), or 1,N6-ethenoadenosine (εA). Examination of conformational changes in DNA clearly revealed multistep DNA rearrangements during the formation of the catalytic complex. These structural rearrangements of DNA are directly associated with the capacity of damaged DNA for enzyme-induced bending and unwinding, which are required for eversion of the damaged nucleotide from the DNA duplex and for its placement into the active site of the enzyme. Taken together, the results experimentally prove the factors that control substrate specificity of the AP endonuclease zAPE1.

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85165956207&origin=inward&txGid=79b7192bb35d0ebcbeeaebd72adc5d17

U2 - 10.3390/ijms241411474

DO - 10.3390/ijms241411474

M3 - Article

C2 - 37511233

VL - 24

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 14

M1 - 11474

ER -