Uracil-DNA glycosylase: Structural, thermodynamic and kinetic aspects of lesion search and recognition

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

doi:10.1016/j.mrfmmm.2009.10.017

Cited by 97 publications

(161 citation statements)

References 94 publications

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“…Observations that protein-DNA nonspecific complexes have relative orientations similar to the corresponding specific complexes (31)(32)(33)(34)(35) suggest that nonspecifically-bound proteins diffuse along a helical path, e.g., the major groove. This restriction will reduce the rate constant k 3 for nonspecific binding and increase the rate constant κ 3− for escape to the bulk solution.…”

Section: Resultsmentioning

confidence: 99%

Rapid search for specific sites on DNA through conformational switch of nonspecifically bound proteins

Zhou

2011

Proc. Natl. Acad. Sci. U.S.A.

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We develop a theory for the rapid search of specific sites on DNA, via a mechanism in which a nonspecifically-bound protein can switch between two conformations. In the "inactive" conformation, the bound protein has favorable, nonspecific interactions with the DNA, but cannot be recognized by the target site. In the "active" conformation, the protein is recognized by the target site but has a very rugged energy surface elsewhere on the DNA. The rate constant for protein binding to the specific site is calculated by an approach in which the protein, after reaching the DNA surface via 3D diffusion, searches for the target site via 1D diffusion while being allowed to escape to the bulk solution. Mindful of the pitfalls of many previous approximate treatments, we validate our approach against a rigorous solution of the problem when the protein has a fixed conformation. In the 1D diffusion toward the specific site, a conformationally switchable protein predominantly adopts the inactive conformation due to the favorable nonspecific interactions with the DNA, thus maximizing the 1D diffusion constant and minimizing the chance of escape to the bulk solution. Once at the target site, a transition to the active conformation allows the protein to be captured. This induced-switch mechanism provides robust speedup of protein-DNA binding rates, and appears to be adopted by many transcription factors and DNA-modifying enzymes.conformational change | nonspecific binding | binding kinetics E ver since the first demonstration that proteins can bind to specific DNA sequences (1, 2), numerous studies have been carried out to address the question of how a protein can readily find a short specific site on a long . It is widely accepted that the search is accomplished by coupled 3D diffusion in the bulk solution and 1D diffusion, while specifically bound, along the DNA surface. Many theoretical models have focused on the 1D diffusion. Appealing terms such as hopping and jumping have been used to treat excursions into the bulk solution. However, the lack of rigor in previous treatments has led to conflicting results. In particular, whether 1D diffusion can play a significant rate-enhancement role under physiological conditions has been questioned (15). Here we present an approach for calculating the protein-DNA binding rate constant k a that allows for proper coupling between 3D and 1D diffusion. We use this approach to treat the conformational switch of a nonspecifically bound protein, and demonstrate that it provides a robust mechanism for speeding up the search of specific sites.The bimolecular rate constant k a can be rigorously determined from the equation governing the relative translational diffusion, rotational diffusion, and internal motions of the two binding molecules (21). This governing equation involves the potential of mean force in these degrees of freedom and parameters characterizing the external and internal dynamics, such as a position-dependent diffusion constant for the translational diffusion and transition rates...

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Section: Resultsmentioning

confidence: 99%

Rapid search for specific sites on DNA through conformational switch of nonspecifically bound proteins

Zhou

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Zirconium dioxide induced UNG, which eliminates uracil from DNA molecules by cleaving the N-glycosylic bond and initiates the base-excision repair (BER) pathway. Uracil appearing in DNA, for example as a result of cytosine deamination, is potentially mutagenic and deleterious for cell regulation (41).…”

Section: Discussionmentioning

confidence: 99%

Induction of altered mRNA expression profiles caused by fibrous and granular dust

Helmig

Dopp

Wenzel

et al. 2013

Molecular Medicine Reports

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“…2 is a member of the uracil DNA glycosylase family of enzymes, which initiate base excision repair (BER) of uracil that results from deamination of cytosine or the misincorporation of uracil or other thymidine analogs during DNA replication (1)(2)(3)(4). Importantly, UNG is the main glycosylase that removes uracil and 5-fluorouracil lesions that accumulate in DNA following treatment of cancers with the closely related fluoropyrimidine anticancer agents 5-fluorouracil and 5-fluorodeoxyuridine (floxuridine) (5)(6)(7).…”

Section: Uracil N-glycosylase (Ung)mentioning

confidence: 99%

Glycogen Synthase Kinase 3 (GSK-3)-mediated Phosphorylation of Uracil N-Glycosylase 2 (UNG2) Facilitates the Repair of Floxuridine-induced DNA Lesions and Promotes Cell Survival

Baehr

Huntoon

Hoang

et al. 2016

Journal of Biological Chemistry

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Edited by Patrick SungUracil N-glycosylase 2 (UNG2), the nuclear isoform of UNG, catalyzes the removal of uracil or 5-fluorouracil lesions that accumulate in DNA following treatment with the anticancer agents 5-fluorouracil and 5-fluorodeoxyuridine (floxuridine), a 5-fluorouracil metabolite. By repairing these DNA lesions before they can cause cell death, UNG2 promotes cancer cell survival and is therefore critically involved in tumor resistance to these agents. However, the mechanisms by which UNG2 is regulated remain unclear. Several phosphorylation sites within the N-terminal regulatory domain of UNG2 have been identified, although the effects of these modifications on UNG2 function have not been fully explored, nor have the identities of the kinases involved been determined. Here we show that glycogen synthase kinase 3 (GSK-3) interacts with and phosphorylates UNG2 at Thr 60 and that Thr 60 phosphorylation requires a Ser 64 priming phosphorylation event. We also show that mutating Thr 60 or Ser 64 to Ala increases the half-life of UNG2, reduces the rate of in vitro uracil excision, and slows UNG2 dissociation from chromatin after DNA replication. Using an UNG2-deficient ovarian cancer cell line that is hypersensitive to floxuridine, we show that GSK-3 phosphorylation facilitates UNG2-dependent repair of floxuridine-induced DNA lesions and promotes tumor cell survival following exposure to this agent. These data suggest that GSK-3 regulates UNG2 and promotes DNA damage repair.2 is a member of the uracil DNA glycosylase family of enzymes, which initiate base excision repair (BER) of uracil that results from deamination of cytosine or the misincorporation of uracil or other thymidine analogs during DNA replication (1-4). Importantly, UNG is the main glycosylase that removes uracil and 5-fluorouracil lesions that accumulate in DNA following treatment of cancers with the closely related fluoropyrimidine anticancer agents 5-fluorouracil and 5-fluorodeoxyuridine (floxuridine) (5-7). Consistent with this observation, we and others have shown previously that UNG protects cells from death induced by agents that cause incorporation of uracil or 5-fluorouracil during DNA replication, likely by removing these lesions before they can cause cell death (4, 6 -8).Mammalian cells express two isoforms of UNG: UNG1, a mitochondrial isoform, and UNG2, a nuclear isoform. Both isoforms have identical C-terminal catalytic domains but differ in the N-terminal regulatory domain because of differential promoter usage and alternative splicing (9). These isoforms are expressed highly during late G 1 /early S phase of the cell cycle and are reduced in late S phase through proteasome-mediated degradation (10, 11).UNG initiates BER by detecting and excising the damaged base, leaving an apyrimidinic site, which recruits an apurinic/ apyrimidinic endonuclease (12) to cleave the DNA backbone 5Ј to the damaged base. The single-stranded nick is then repaired through the subsequent activity of a DNA polymerase and DNA ligase. Although BER can be...

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Uracil-DNA glycosylase: Structural, thermodynamic and kinetic aspects of lesion search and recognition

Cited by 97 publications

References 94 publications

Rapid search for specific sites on DNA through conformational switch of nonspecifically bound proteins

Rapid search for specific sites on DNA through conformational switch of nonspecifically bound proteins

Induction of altered mRNA expression profiles caused by fibrous and granular dust

Glycogen Synthase Kinase 3 (GSK-3)-mediated Phosphorylation of Uracil N-Glycosylase 2 (UNG2) Facilitates the Repair of Floxuridine-induced DNA Lesions and Promotes Cell Survival

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