TY - JOUR

T1 - Randomization of the Damaged Base-Capping Loop Rewires Substrate Specificity in Formamidopyrimidine–DNA Glycosylase

AU - Bulgakov, Nikita A.

AU - Amanova, Margarita M.

AU - Endutkin, Anton V.

AU - Makasheva, Kristina A.

AU - Coste, Franck

AU - Castaing, Bertrand

AU - Golyshev, Victor M.

AU - Lomzov, Alexander A.

AU - Yudkina, Anna V.

AU - Zharkov, Dmitry O.

N1 - Nikita A. Bulgakov, Margarita M. Amanova, Anton V. Endutkin, Kristina A. Makasheva, Franck Coste, Bertrand Castaing, Victor M. Golyshev, Alexander A. Lomzov, Anna V. Yudkina, Dmitry O. Zharkov; Randomization of the Damaged Base-Capping Loop Rewires Substrate Specificity in Formamidopyrimidine–DNA Glycosylase. Biochemistry 3 November 2026; 65 (21): 3419–3433. https://doi.org/10.1021/acs.biochem.6c00579 This research was supported by the Russian Science Foundation (grant 21-64-00017p). Partial salary support from the Russian Ministry of Science and Higher Education (grant 125012300657-2) is acknowledged. F.C. and B.C. were funded by a grant from the Ligue contre le Cancer du GrandOuest [committees 37, 41, and 45]. We thank Drs Murat Saparbaev and Alexandre Ishchenko for their valuable discussion.

PY - 2026/10/16

Y1 - 2026/10/16

N2 - Formamidopyrimidine–DNA glycosylase (Fpg) is a bacterial base excision repair enzyme that removes 8-oxoguanine (8-oxoG) and some other oxidatively damaged nucleobases from DNA. Fpg hydrolyzes the N-glycosidic bond of the damaged nucleotide and nicks DNA at the resulting apurinic/apyrimidinic (AP) site by β-elimination. Together with bacterial endonuclease VIII and eukaryotic homologues, more specific for damaged pyrimidines, Fpg belongs to a helix–two-turn–helix (H2TH) superfamily of DNA glycosylases. Crystal structures of Fpg reveal that the enzyme recognizes 8-oxoG by flipping it out of the DNA duplex and capping it with a flexible tetrapeptide loop. However, this loop is poorly conserved and contacts 8-oxoG only through the main-chain amides, raising questions about the enzyme specificity determinants. To clarify the mechanism of 8-oxoG recognition, we have produced and characterized five variants of Fpg from Lactococcus lactis with a randomized capping loop. All of these mutants were deficient in 8-oxoG repair, both in vitro and in the E. coli complementation assay, yet retained the ability to bind AP site-containing DNA, form a Schiff base covalent reaction intermediate, and catalyze β-elimination. The mutants were still active in the removal of 5-hydroxyuracil (OHU), an alternative Fpg substrate. All loop mutants showed relaxed opposite-base specificity, processing AP sites and OHU opposite either C or A. Thus, whereas the general architecture of the active site in H2TH DNA glycosylases is permissive for the excision of a wide range of oxidized bases, the capping loop endows Fpg with the specificity for its biologically relevant substrate, 8-oxoguanine.

AB - Formamidopyrimidine–DNA glycosylase (Fpg) is a bacterial base excision repair enzyme that removes 8-oxoguanine (8-oxoG) and some other oxidatively damaged nucleobases from DNA. Fpg hydrolyzes the N-glycosidic bond of the damaged nucleotide and nicks DNA at the resulting apurinic/apyrimidinic (AP) site by β-elimination. Together with bacterial endonuclease VIII and eukaryotic homologues, more specific for damaged pyrimidines, Fpg belongs to a helix–two-turn–helix (H2TH) superfamily of DNA glycosylases. Crystal structures of Fpg reveal that the enzyme recognizes 8-oxoG by flipping it out of the DNA duplex and capping it with a flexible tetrapeptide loop. However, this loop is poorly conserved and contacts 8-oxoG only through the main-chain amides, raising questions about the enzyme specificity determinants. To clarify the mechanism of 8-oxoG recognition, we have produced and characterized five variants of Fpg from Lactococcus lactis with a randomized capping loop. All of these mutants were deficient in 8-oxoG repair, both in vitro and in the E. coli complementation assay, yet retained the ability to bind AP site-containing DNA, form a Schiff base covalent reaction intermediate, and catalyze β-elimination. The mutants were still active in the removal of 5-hydroxyuracil (OHU), an alternative Fpg substrate. All loop mutants showed relaxed opposite-base specificity, processing AP sites and OHU opposite either C or A. Thus, whereas the general architecture of the active site in H2TH DNA glycosylases is permissive for the excision of a wide range of oxidized bases, the capping loop endows Fpg with the specificity for its biologically relevant substrate, 8-oxoguanine.

UR - https://www.mendeley.com/catalogue/58fdaa8e-cea8-323a-8d7d-8965654977e0/

UR - https://www.scopus.com/pages/publications/105055323255

U2 - 10.1021/acs.biochem.6c00579

DO - 10.1021/acs.biochem.6c00579

M3 - Article

C2 - 42975162

VL - 65

SP - 3419

EP - 3433

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

ER -