Ionic strength and magnesium affect the specificity of <i>Escherichia coli</i> and human 8‐oxoguanine‐DNA glycosylases

Sidorenko, Viktoriya S.; Mechetin, Grigory V.; Nevinsky, Georgy A.; Zharkov, Dmitry O.

doi:10.1111/j.1742-4658.2008.06521.x

Cited by 8 publications

(8 citation statements)

References 70 publications

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“…5 and Supporting Fig. S6, wild-type OGG efficiently formed a complex with THF-DNA (Kd = 0.14 ± 0.03 μM), with the affinity within the range of values reported in the literature (tens to hundreds nM) that were determined by the gel shift assay and fluorescence titration (49,(53)(54)(55). For I145M, R161W, and S292N we could not reach binding saturation, but their estimated Kd values were 7-10-fold higher (0.90 ± 0.24 μM, 1.06 ± 0.51 μM, and 1.39 ± 0.45 μM, respectively) than that of the wild-type.…”

Section: Biochemical Characterization Of Ogg1 Variantssupporting

confidence: 74%

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity

Popov

Endutkin

Yatsenko

et al. 2021

Journal of Biological Chemistry

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DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important systems. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemotherapy and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present, they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structures. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine DNA glycosylase. In parallel, we have experimentally characterized the activity, thermostability, and DNA binding in a subset of these mutant proteins. Among the analyzed variants of 8-oxoguanine DNA glycosylase, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.

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Section: Biochemical Characterization Of Ogg1 Variantssupporting

confidence: 74%

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity

Popov

Endutkin

Yatsenko

et al. 2021

Journal of Biological Chemistry

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“…This may cause some DNA-dependent enzymes to be much more sensitive to the concentration of Mg 2+ than of monovalent cations even if they, like DNA glycosylases, do not require Mg 2+ for their activity [24]. Therefore, it was interesting to compare the effect of Mg 2+ and K + on the processivity of Ung.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of translocation of uracil–DNA glycosylase from Escherichia coli between distributed lesions

Mechetin

Zharkov

2011

Biochemical and Biophysical Research Communications

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Uracil-DNA glycosylase (Ung) is a DNA repair enzyme that excises uracil bases from DNA, where they appear through deamination of cytosine or incorporation from a cellular dUTP pool. DNA repair enzymes often use one-dimensional diffusion along DNA to accelerate target search; however, this mechanism remains poorly investigated mechanistically. We used oligonucleotide substrates containing two uracil residues in defined positions to characterize one-dimensional search of DNA by Escherichia coli Ung. Mg2+ ions suppressed the search in double-stranded DNA to a higher extent than K+ likely due to tight binding of Mg2+ to DNA phosphates. Ung was able to efficiently overcome short single-stranded gaps within double-stranded DNA. Varying the distance between the lesions and fitting the data to a theoretical model of DNA random walk, we estimated the characteristic one-dimensional search distance of ~100 nucleotides and translocation rate constant of ~2×106 s−1.

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“… (A) Localization of the mutated residues in the three‐dimensional structure of OGG1 (Protein Data Bank reference number: 1EBM [46]). The DNA is shown as a stick model, and the protein as a cartoon.…”

Section: Resultsmentioning

confidence: 99%

“…Figure 2 shows a typical dependence of the reaction velocity on the substrate concentration in double reciprocal coordinates for the wild‐type enzyme. The specificity constant, k sp = k cat / K m , was calculated for each enzyme and substrate, and the ratio of the k sp for 8‐oxoGua:Cyt to the k sp for 8‐oxoGua:Ade was used as a measure of the biologically relevant opposite‐base specificity (C/A specificity) [46]. In the wild‐type enzyme, the C/A specificity of 4.9 was due mostly to the lower value of K m for the 8‐oxoGua:Cyt substrate (Tables 2 and 3), similar to what was reported in the literature [23,45].…”

Section: Resultsmentioning

confidence: 99%

“…The C/A specificity of OGG1 is important in preventing 8‐oxoGua‐induced mutagenesis. We have shown that the C/A specificity of OGG1 and Fpg is highest under nearly physiological conditions, owing to a sharp decrease in the enzyme’s activity on 8‐oxoGua:Ade substrates with increasing ionic strength and Mg 2+ concentration [46], and that APEX1 stimulates OGG1 to a higher degree on 8‐oxoGua:Cyt than on 8‐oxoGua:Ade substrates [24]. In comparison with these factors, the natural variations and phosphomimetic mutations in OGG1 had a lower impact on the C/A specificity, which varied between 70% and 240% of the specificity of the wild‐type enzyme.…”

Section: Discussionmentioning

confidence: 99%

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Substrate specificity and excision kinetics of natural polymorphic variants and phosphomimetic mutants of human 8‐oxoguanine‐DNA glycosylase

et al. 2009

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Human 8-oxoguanine-DNA glycosylase (OGG1) efficiently removes mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine when paired with cytosine in oxidatively damaged DNA. Excision of 8-oxoGua mispaired with adenine may lead to G fi T transversions. Post-translational modifications such as phosphorylation could affect the cellular distribution and enzymatic activity of OGG1. Mutations and polymorphisms of OGG1 may affect the enzymatic activity and have been associated with increased risk of several cancers. In this study, we used double-stranded oligodeoxynucleotides containing 8-oxoGua:Cyt or 8-oxoGua:Ade pairs, as well as c-irradiated calf thymus DNA, to investigate the kinetics and substrate specificity of several known OGG1 polymorphic variants and phosphomimetic Ser fi Glu mutants. Among the polymorphic variants, A288V and S326C displayed opposite-base specificity similar to that of wild-type OGG1, whereas OGG1-D322N was 2.3-fold more specific for the correct opposite base than the wild-type enzyme. All phosphomimetic mutants displayed 1.5-3-fold lower ability to remove 8-oxoGua in both assays, whereas the substrate specificity of the phosphomimetic mutants was similar to that of the wild-type enzyme. OGG1-S326C efficiently excised 8-oxoGua from oligodeoxynucleotides and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from c-irradiated DNA, but excised 8-oxoG rather inefficiently from c-irradiated DNA. Otherwise, k cat values for 8-oxoGua excision obtained from both types of experiments were similar for all OGG1 variants studied. It is known that the human AP endonuclease APEX1 can stimulate OGG1 activity by increasing its turnover rate. However, when wild-type OGG1 was replaced by one of the phosphomimetic mutants, very little stimulation of 8-oxoGua removal was observed in the presence of APEX1.Abbreviations 8-oxoGua, 8-oxo-7,8-dihydroguanine; AP, apurinic ⁄ apyrimidinic; BER, base excision repair; CDK4, cyclin-dependent kinase 4; FapyAde, 4,6-diamino-5-formamidopyrimidine; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; OGG1, 8-oxoguanine-DNA glycosylase; PKC, protein kinase C.

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Ionic strength and magnesium affect the specificity of Escherichia coli and human 8‐oxoguanine‐DNA glycosylases

Cited by 8 publications

References 70 publications

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity

Mechanism of translocation of uracil–DNA glycosylase from Escherichia coli between distributed lesions

Substrate specificity and excision kinetics of natural polymorphic variants and phosphomimetic mutants of human 8‐oxoguanine‐DNA glycosylase

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