Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

Kuznetsov, Nikita А.; Kladova, Olga A.; Кузнецова, А. А.; Ищенко, А. А.; Saparbaev, Murat; Zharkov, Dmitry O.; Fedorova, Olga S.

doi:10.1074/jbc.m114.621128

Cited by 45 publications

(36 citation statements)

References 63 publications

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“…It is known that the fluorescence intensity of aPu in DNA depends on the fluorophore microenvironment changes, for example, as a result of local melting of the duplex in the immediate vicinity of the fluorophore [24-26]. In double-stranded DNA structures, during stacking interaction with neighboring bases, the fluorescence intensity of aPu residues reduces significantly in comparison with single-stranded DNA.…”

Section: Resultsmentioning

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: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…Recent crystallographic [36, 37], kinetics [10, 38] and single molecule studies [9, 11] provide strong evidence that DNA glycosylases locate damaged DNA bases by one-dimensional diffusion along the DNA helix that is random, bidirectional, and is consistent with tracking rotationally along the DNA backbone. The Nth glycosylases rely upon the insertion of three amino acids into the DNA helix to stabilize the duplex upon eversion of the damaged base into the enzyme’s active site pocket and in EcoNth, these residues are Ile79, Leu81, and Gln41.…”

Section: Discussionmentioning

confidence: 99%

“…This mechanism provides an explanation for how the Nth search might be independent of specific interactions within the binding pocket, allowing for recognition of a diverse set of substrates. A recent stopped-flow kinetics analysis showed Nth to induce several fast sequential conformational changes in DNA during binding, lesion recognition and forming the catalytically competent structure and that the first phase of non-specific binding may be insertion of Leu81 [10]. Of note, the EcoNth Leu81 residue counterpart in hNTHL1 is Phe181, which is bulkier, and may “sense” aromatic structures.…”

Section: Discussionmentioning

confidence: 99%

Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues

et al. 2017

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The base excision repair DNA glycosylases, EcoNth and hNTHL1, are homologous, with reported overlapping yet different substrate specificities. The catalytic amino acid residues are known and are identical between the two enzymes although the exact structures of the substrate binding pockets remain to be determined. We sought to explore the sequence basis of substrate differences using a phylogeny-based design of site-directed mutations. Mutations were made for each enzyme in the vicinity of the active site and we examined these variants for glycosylase and lyase activity. Single turnover kinetics were done on a subgroup of these, comparing activity on two lesions, 5,6-dihydrouracil and 5,6-dihydrothymine, with different opposite bases. We report that wild type hNTHL1 and EcoNth are remarkably alike with respect to the specificity of the glycosylase reaction, and although hNTHL1 is a much slower enzyme than EcoNth, the tighter binding of hNTHL1 compensates, resulting in similar kcat/Kd values for both enzymes with each of the substrates tested. For the hNTHL1 variant Gln287Ala, the specificity for substrates positioned opposite G is lost, but not that of substrates positioned opposite A, suggesting a discrimination role for this residue. The EcoNth Thr121 residue influences enzyme binding to DNA, as binding is significantly reduced with the Thr121Ala variant. Finally, we present evidence that hNTHL1 Asp144, unlike the analogous EcoNth residue Asp44, may be involved in resolving the glycosylase transition state.

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“…We substituted alanine for the wedge residue in each of these glycosylases, and in each case, the variant enzyme scanned along undamaged DNA at a significantly faster rate than the wild-type enzyme (Figure 3). In further support of the amino acid wedge being responsible for the lesion search, (Kuznetsov et al, 2015b) recently showed, using stopped flow kinetics, that the first phase of nonspecific binding of Nth to a lesion may be insertion of Leu81, its wedge residue.…”

Section: The Target Search On Undamaged Dnamentioning

confidence: 94%

Visualizing the search for radiation-damaged DNA bases in real time

Lee

Wallace

2016

Radiation Physics and Chemistry

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The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

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Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

Cited by 45 publications

References 63 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

Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues

Visualizing the search for radiation-damaged DNA bases in real time

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