Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1

Кузнецова, А. А.; Kuznetsov, Nikita А.; Ищенко, А. А.; Saparbaev, Murat; Fedorova, Olga S.

doi:10.1016/j.bbagen.2014.07.016

Cited by 28 publications

(25 citation statements)

References 70 publications

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“…The catalytic domain of human DNA glycosylase, MBD4 cat (amino acid residues 426–580), was isolated from the cells of Escherichia coli Rosetta 2 transformed with the plasmid pET29b-MBD4cat as described previously [11, 14]. The plasmid pET29b-MBD4 cat containing the MBD4 cat gene was kindly provided by M.K.…”

Section: Methodsmentioning

confidence: 99%

Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4

et al. 2017

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The MBD4 enzyme initiates the process of DNA demethylation by the excision of modified DNA bases, resulting in the formation of apurinic/apyrimidinic sites. MBD4 contains a methyl-CpG-binding domain which provides the localization of the enzyme at the CpG sites, and a DNA glycosylase domain that is responsible for the catalytic activity. The aim of this work was to clarify the mechanisms of specific site recognition and formation of catalytically active complexes between model DNA substrates and the catalytic N-glycosylase domain MBD4cat. The conformational changes in MBD4cat and DNA substrates during their interaction were recorded in real time by stopped-flow detection of the fluorescence of tryptophan residues in the enzyme and fluorophores in DNA. A kinetic scheme of MBD4cat interaction with DNA was proposed, and the rate constants for the formation and decomposition of transient reaction intermediates were calculated. Using DNA substrates of different lengths, the formation of the catalytically active complex was shown to follow the primary DNA binding step which is responsible for the search and recognition of the modified base. The results reveal that in the primary complex of MBD4cat with DNA containing modified nucleotides, local melting and bending of the DNA strand occur. On the next step, when the catalytically competent conformation of the enzyme-substrate complex is formed, the modified nucleotide is everted from the double DNA helix into the active center and the void in the helix is filled by the enzyme’s amino acids.

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Section: Methodsmentioning

confidence: 99%

Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4

et al. 2017

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“…The AP-site is chemically unstable; therefore, in many studies, its stable analog (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F-site) has been successfully used. [49][50][51] To detect the conformational changes of hSMUG1 during binding of an abasic DNA product we also used the DNA ligand containing an F-site. The interaction of the enzyme with the F-ligand led to a biphasic change in the Trp fluorescence intensity (Fig.…”

Section: Interaction Of Hsmug1 With Undamaged Dnamentioning

confidence: 99%

Pre-steady-state kinetic analysis of damage recognition by human single-strand selective monofunctional uracil-DNA glycosylase SMUG1

et al. 2017

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In all organisms, DNA glycosylases initiate base excision repair pathways resulting in removal of aberrant bases from DNA. Human SMUG1 belongs to the superfamily of uracil-DNA glycosylases catalyzing the hydrolysis of the N-glycosidic bond of uridine and uridine lesions bearing oxidized groups at C5: 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), and 5-hydroxyuridine (5hoU). An apurinic/apyrimidinic (AP) site formed as the product of an N-glycosylase reaction is tightly bound to hSMUG1, thus inhibiting the downstream action of AP-endonuclease APE1. The steady-state kinetic parameters (k and K; obtained from the literature) correspond to the enzyme turnover process limited by the release of hSMUG1 from the complex with the AP-site. In the present study, our objective was to carry out a stopped-flow fluorescence analysis of the interaction of hSMUG1 with a DNA substrate containing a dU:dG base pair to follow the pre-steady-state kinetics of conformational changes in both molecules. A comparison of kinetic data obtained by means of Trp and 2-aminopurine fluorescence and Förster resonance energy transfer (FRET) detection allowed us to elucidate the stages of specific and nonspecific DNA binding, to propose the mechanism of damaged base recognition by hSMUG1, and to determine the true rate of the catalytic step. Our results shed light on the kinetic mechanism underlying the initiation of base excision repair by hSMUG1 using the "wedge" strategy for DNA lesion search.

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“…The method for isolation of wild-type APE1 was described previously. 29,31 The protein concentration was measured by the Bradford method; 32 the stock solution was stored at À20 1C.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1

et al. 2016

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Here, we used stopped-flow fluorescence techniques to conduct a comparative kinetic analysis of the conformational transitions in human apurinic/apyrimidinic endonuclease 1 (APE1) and in DNA containing an abasic site in the course of their interaction. Effects of monovalent (K(+)) and divalent (Mg(2+), Mn(2+), Ca(2+), Zn(2+), Cu(2+), and Ni(2+)) metal ions on DNA binding and catalytic stages were studied. It was shown that the first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreased at a high concentration (5-250 mM) of monovalent K(+) ions, indicating the involvement of electrostatic interactions in this stage. It was also shown that Cu(2+) ions abrogated the DNA binding ability of APE1, possibly, due to a strong interaction with DNA bases and the sugar-phosphate backbone. In the case of Ca(2+) ions, the catalytic activity of APE1 was lost completely with retention of binding potential. Thus, the enzymatic activity of APE1 is increased in the order Zn(2+) < Ni(2+) < Mn(2+) < Mg(2+). Circular dichroism spectra and calculation of the contact area between APE1 and DNA reveal that Mg(2+) ions stabilize the protein structure and the enzyme-substrate complex.

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Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1

Cited by 28 publications

References 70 publications

Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4

Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4

Pre-steady-state kinetic analysis of damage recognition by human single-strand selective monofunctional uracil-DNA glycosylase SMUG1

Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1

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