TY - JOUR

T1 - Molecular dynamics simulation of the opposite-base preference and interactions in the active site of formamidopyrimidine-DNA glycosylase

AU - Popov, Alexander V.

AU - Endutkin, Anton V.

AU - Vorobjev, Yuri N.

AU - Zharkov, Dmitry O.

N1 - Publisher Copyright: © 2017 The Author(s).

PY - 2017/5/8

Y1 - 2017/5/8

N2 - Background: Formamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1′ of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg-DNA complexes have been solved, yet no structure with A opposite the lesion is available. Results: Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1-Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein-DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition. Conclusion: Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.

AB - Background: Formamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1′ of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg-DNA complexes have been solved, yet no structure with A opposite the lesion is available. Results: Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1-Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein-DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition. Conclusion: Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.

KW - 8-oxoguanine

KW - DNA glycosylase

KW - Fpg

KW - Molecular dynamics

KW - Opposite-base specificity

KW - Reaction mechanism

KW - Catalytic Domain

KW - Molecular Dynamics Simulation

KW - Guanine/analogs & derivatives

KW - DNA-Formamidopyrimidine Glycosylase/chemistry

KW - DNA/chemistry

KW - Base Pairing

KW - Lactococcus lactis/enzymology

KW - ESCHERICHIA-COLI FPG

KW - STRUCTURAL INSIGHTS

KW - OXIDATIVELY DAMAGED DNA

KW - REPAIR ENZYME

KW - CRYSTAL-STRUCTURE

KW - GRAPHICAL USER-INTERFACE

KW - EXCISION-REPAIR

KW - BIOMOLECULAR SIMULATIONS

KW - SUBSTRATE-SPECIFICITY

KW - SEQUENCE ALIGNMENTS

UR - http://www.scopus.com/inward/record.url?scp=85018411806&partnerID=8YFLogxK

U2 - 10.1186/s12900-017-0075-y

DO - 10.1186/s12900-017-0075-y

M3 - Article

C2 - 28482831

AN - SCOPUS:85018411806

VL - 17

SP - 1

EP - 19

JO - BMC Structural Biology

JF - BMC Structural Biology

SN - 1472-6807

IS - 1

M1 - 5

ER -