Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

Kuznetsov, Nikita А.; Bergonzo, Christina; Campbell, Arthur J.; Li, Haoquan; Mechetin, Grigory V.; Santos, Carlos de los; Grollman, Arthur P.; Fedorova, Olga S.; Zharkov, Dmitry O.; Simmerling, Carlos

doi:10.1093/nar/gku1300

Cited by 52 publications

(61 citation statements)

References 48 publications

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“…This is also consistent with our previous work showing that buckling induced by the wedge intercalation in Fpg destabilizes the intrahelical state. 38 The energetic cost of breaking the G:C or 8-oxoG:C pairs are comparable, indicating no preferential opening of damaged DNA. Notably, the energy cost for the 8-oxoG:C opening in OGG1 is comparable to that in Fpg, 19 and also comparable to the energy barriers of OGG1/Fpg sliding along DNA (0.5 to 2 kcal/mol), 5 but is significantly lower than G opening in the context of B-DNA, which has a ~10 kcal/mol barrier.…”

Section: Resultsmentioning

confidence: 99%

DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase

Li¹,

Endutkin

Bergonzo³

et al. 2017

J. Am. Chem. Soc.

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8-Oxoguanine (8-oxoG), a mutagenic DNA lesion generated under oxidative stress, differs from its precursor guanine by only two substitutions (O8 andH7). Human 8-oxoguanine glycosylase 1 (OGG1) can locate and remove 8-oxoG through extrusion and excision. To date, it remains unclear how OGG1 efficiently distinguishes 8-oxoG from a large excess of undamaged DNA bases. We recently showed that formamidopyrimidine–DNA glycosylase (Fpg), a bacterial functional analog of OGG1, can selectively facilitate eversion of oxoG by stabilizing several intermediate states, and it is intriguing whether OGG1 also employs a similar mechanism in lesion recognition. Here, we use molecular dynamics simulations to explore the mechanism by which OGG1 discriminates between 8-oxoG and guanine along the base eversion pathway. The MD results suggest an important role for kinking of the DNA by the glycosylase, which positions DNA phosphates in a way that assists lesion recognition during base eversion. The computational predictions were validated through experimental enzyme assays on phosphorothioate substrate analogs. Our simulations suggest that OGG1 distinguishes between 8-oxoG and G using their chemical dissimilarities not only at the active site, but also at earlier stages during base eversion, and this mechanism is at least partially conserved in Fpg despite lack of structural homology. The similarity also suggests that lesion recognition through multiple gating steps may be a common theme in DNA repair. Our results provide new insight into how enzymes can exploit kinetics and DNA conformational changes to probe the chemical modifications present in DNA lesions.

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Section: Resultsmentioning

confidence: 99%

DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase

Li¹,

Endutkin

Bergonzo³

et al. 2017

J. Am. Chem. Soc.

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“…8). As in the case of previously studied DNA glycosylases, E. coli Fpg (28,29,31,63) and human OGG1 (20,21), binding of Endo III to DNA begins most likely with an insertion of the wedge amino acids, Gln 41 and Leu 81 , into the helix (61,62). Insertion of other amino acids and eversion of the damaged nucleotide into the active site proceed at the next stage of specific lesion recognition, resulting in "stapling" of the enzyme on DNA.…”

Section: Resultsmentioning

confidence: 90%

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

Kuznetsov

Kladova

Кузнецова

et al. 2015

Journal of Biological Chemistry

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Background: Endonuclease III is responsible for base excision repair of oxidized or reduced pyrimidine bases. Results: Stopped-flow kinetics analysis of endonuclease III interaction with DNA was performed. Conclusion: Endonuclease III uses a multistep mechanism of damage recognition, which likely involves Gln 41 and Leu 81 as lesion sensors. Significance: The results provide new insight into the mechanism of damage recognition by DNA glycosylases of the helixhairpin-helix-GPD structural superfamily.

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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%

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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Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

Cited by 52 publications

References 48 publications

DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase

DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

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

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