Research output: Contribution to journal › Article › peer-review
Randomization of the Damaged Base-Capping Loop Rewires Substrate Specificity in Formamidopyrimidine–DNA Glycosylase. / Bulgakov, Nikita A.; Amanova, Margarita M.; Endutkin, Anton V. et al.
In: Biochemistry, Vol. 65, 16.10.2026, p. 3419-3433.Research output: Contribution to journal › Article › peer-review
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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 -
ID: 84986130