TY - JOUR

T1 - Unique structural mode for opposite-base tolerance in endonuclease VIII

AU - Bulgakov, Nikita A.

AU - Endutkin, Anton V.

AU - MacCarthy, Christopher

AU - Shevtsov, Mikhail B.

AU - Kapranov, Ivan A.

AU - Yudkina, Anna V.

AU - Borshchevskiy, Valentin I.

AU - Zharkov, Dmitry O.

N1 - Nikita A. Bulgakov, Anton V. Endutkin, Christopher MacCarthy, Mikhail B. Shevtsov, Ivan A. Kapranov, Anna V. Yudkina, Valentin I. Borshchevskiy, Dmitry O. Zharkov, Unique structural mode for opposite-base tolerance in endonuclease VIII, DNA Repair, Volume 167, 2026, 103962, ISSN 1568-7864, https://doi.org/10.1016/j.dnarep.2026.103962. We gratefully acknowledge the Shanghai Synchrotron Radiation Facility (SSRF) for providing beamtime at BL02U1 and its staff for their assistance during data collection. This research was supported by the Russian Science Foundation (grant 21–64–00017p, structure solving). Partial support from the Russian Ministry of Science and Higher Education is acknowledged (project 125012300657–2, biochemical experiments, and agreement 075–03–2025–662, project FSMG-2024–0012, crystallization and X-ray data collection).

PY - 2026/11

Y1 - 2026/11

N2 - Endonuclease VIII (Nei) is a DNA repair enzyme with broad substrate specificity for oxidative lesions, excising damaged bases paired with any normal base. The structural basis for this opposite-base tolerance is unknown, since only the structure of the Nei–DNA complex with A opposite the lesion had been determined. Here we address this question by solving crystal structures of Escherichia coli Nei conjugates with DNA duplexes containing C, G, or T opposite the lesion. While estranged purine bases are recognized by Nei via a non-specific hydrogen bond to N3, pyrimidine bases undergo a 180° rotation into the syn conformation. In this unique orientation, their Watson–Crick edge faces the major groove, forming no specific contacts with Nei. Substrates with 5-hydroxyuracil opposite T analogs carrying bulky C5-substitutions are cleaved by Nei less efficiently, consistent with the intrahelical location of the C5–C6 edge of the estranged pyrimidine bases. We have also identified several hydrogen bond networks within the Nei molecule that may facilitate accommodation of different DNA structures and stabilize the catalytic conformation of the enzyme. The mechanism of opposite-base tolerance through pyrimidine flipping is unprecedented among DNA glycosylases and may allow Nei to recognize a wide spectrum of oxidative lesions.

AB - Endonuclease VIII (Nei) is a DNA repair enzyme with broad substrate specificity for oxidative lesions, excising damaged bases paired with any normal base. The structural basis for this opposite-base tolerance is unknown, since only the structure of the Nei–DNA complex with A opposite the lesion had been determined. Here we address this question by solving crystal structures of Escherichia coli Nei conjugates with DNA duplexes containing C, G, or T opposite the lesion. While estranged purine bases are recognized by Nei via a non-specific hydrogen bond to N3, pyrimidine bases undergo a 180° rotation into the syn conformation. In this unique orientation, their Watson–Crick edge faces the major groove, forming no specific contacts with Nei. Substrates with 5-hydroxyuracil opposite T analogs carrying bulky C5-substitutions are cleaved by Nei less efficiently, consistent with the intrahelical location of the C5–C6 edge of the estranged pyrimidine bases. We have also identified several hydrogen bond networks within the Nei molecule that may facilitate accommodation of different DNA structures and stabilize the catalytic conformation of the enzyme. The mechanism of opposite-base tolerance through pyrimidine flipping is unprecedented among DNA glycosylases and may allow Nei to recognize a wide spectrum of oxidative lesions.

KW - DNA repair

KW - Endonuclease VIII

KW - Protein–DNA interactions

KW - Substrate specificity

KW - X-ray structure

KW - Репарация ДНК

KW - Эндонуклеаза VIII

KW - Взаимодействие белок-ДНК

KW - Рентгеновская структура

KW - Субстратная специфичность

UR - https://www.mendeley.com/catalogue/cd8517e4-ac54-334c-8adc-7409821e5c23/

UR - https://www.scopus.com/pages/publications/105051655375

U2 - 10.1016/j.dnarep.2026.103962

DO - 10.1016/j.dnarep.2026.103962

M3 - Article

C2 - 42804861

VL - 167

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

M1 - 103962

ER -