Molecular crowding enhances facilitated diffusion of two human DNA glycosylases

Cravens, Shannen L.; Schonhoft, Joseph; Rowland, Meng M.; Rodriguez, Alyssa A.; Anderson, Breeana G.; Stivers, James T.

doi:10.1093/nar/gkv301

Cited by 43 publications

(119 citation statements)

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“…S1B, Table S1). Further, our previous studies have indicated that the target search time of hOGG1 can be adversely affected by the presence of nonspecific DNA sequences 20 . Thus, we infer that a slow step after the formation of the enzyme-DNA complex and before glycosidic bond cleavage is rate-limiting in the first turnover in presence of excess DNA, but in the presence of excess enzyme, the chemical step is rate-limiting.…”

Section: Discussionmentioning

confidence: 99%

“…Full-length hOGG1 20 , wild type human APE1 21 , Δ49APE1 and the catalytic domain of hUNG 22 were expressed and purified as previously described. TDG was a generous gift of Dr. Alexander Drohat.…”

Section: Methodsmentioning

confidence: 99%

“…Errors were estimated by performing three independent replicates of the titrations. In the case of hUNG, which showed very weak binding to the AP site, the saturating anisotropy value was fixed at a value previously observed in titrations using other salt concentrations where saturation could be achieved 20 . This value was consistent with the extrapolated value predicted from the fit to the data set.…”

Section: Figurementioning

confidence: 99%

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AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

et al. 2017

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A major product of oxidative DNA damage is 8-oxoguanine. In humans, 8-oxoguanine DNA Glycosylase (hOGG1) facilitates removal of these lesions, producing an abasic (AP) site in the DNA that is subsequently incised by AP-endonuclease 1 (APE1). APE1 stimulates turnover of several glycosylases by accelerating rate-limiting product release. However, there have been conflicting accounts of whether hOGG1 follows a similar mechanism. In pre-steady state kinetic measurements, we found that addition of APE1 had no effect on the rapid burst phase of 8-oxoguanine excision by hOGG1, but accelerated steady-state turnover (kcat) by ~10-fold. The stimulation by APE1 required divalent cations, was detectable under multiple turnover conditions using limiting concentrations of APE1, did not require flanking DNA surrounding the hOGG1 lesion site, and occurred efficiently even when the first 49 residues of APE1’s N-terminus was deleted. Stimulation by APE1 does not involve relief from product inhibition because thymine DNA glycosylase (TDG), an enzyme that binds more tightly to AP-sites than hOGG1, could not effectively substitute for APE1. A stimulation mechanism involving stable protein-protein interactions between free APE1 and hOGG1, or the DNA bound forms, was excluded using protein crosslinking assays. The combined results indicate a mechanism where dynamic excursions of hOGG1 from the AP-site allow APE1 to invade the site and rapidly incise the phosphate backbone. This mechanism, which allows APE1 to access the AP-site without forming specific interactions with the glycosylase, is a simple and elegant solution to passing along unstable intermediates in base excision repair.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

et al. 2017

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“…The efficiency of the search process relies on the fraction of the total search time that the enzyme remains in the vicinity of DNA as opposed to diffusing through regions of the nucleus that do not contain potential damage sites. The localization of glycosylases to the vicinity of DNA chains involves both intrinsic properties of the enzyme (such as electrostatic or non-electrostatic interaction energies) 5, 6, 7, 8 , as well as properties of the nuclear environment (such as molecular crowding) that tend to favor the formation of DNA-protein complexes 9, 10, 11, 12, 13 . An important property that affects the search time is the binding lifetime of a DNA glycosylase with undamaged DNA.…”

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confidence: 99%

Comparative Effects of Ions, Molecular Crowding, and Bulk DNA on the Damage Search Mechanisms of hOGG1 and hUNG

Cravens

Stivers

2016

Biochemistry

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The energetic nature of the interactions of DNA base excision repair glycosylases with undamaged and damaged DNA and the nuclear environment are expected to significantly impact the time it takes for these enzymes to search for damaged DNA bases. In particular, the high concentration of monovalent ions, macromolecule crowding, and densely packed DNA chains in the cell nucleus could alter the search mechanisms of these enzymes as compared to findings in dilute buffers typically used in in vitro experiments. Here we utilize an in vitro system where the concerted effects of monovalent ions, macromolecular crowding and high concentrations of bulk DNA chains on the activity of two paradigm human DNA glycosylases can be determined. We find that the energetic nature of the observed binding free energies of human 8-oxoguanine DNA glycosylase (hOGG1) and human uracil DNA glycosylase (hUNG) for undamaged DNA are derived from different sources. While hOGG1 uses primarily non-electrostatic binding interactions with non-specific DNA, hUNG uses a salt-dependent electrostatic binding mode. Both enzymes turn to a non-electrostatic mode in their specific complexes with damaged bases in DNA, which enhances damage site specificity at physiological ion concentrations. Neither enzyme was capable of efficiently locating and removing their respective damaged bases in the combined presence of physiological ions and a bulk DNA chain density approximating that found in the nucleus. However, the addition of an inert crowding agent to mimic macromolecular crowding in the nucleus largely restored their ability to track DNA chains and locate damaged sites. These findings suggest how the concerted action of monovalent ions and crowding could contribute to efficient DNA damage recognition in cells.

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“…The effects of macromolecular crowding on facilitated diffusion, as it relates to more physiological conditions in living cells, have also been recently examined (Elf et al, 2007; Li et al, 2009). The latest experimental (Cravens et al, 2015) and theoretical studies (Brackley et al, 2013; Krepel et al, 2016; Liu and Luo, 2014) have shown that crowding environments can lead to altered balance between three-dimensional and one-dimensional diffusion processes, promoting one-dimensional sliding. While the presence of mobile or immobile obstacles on DNA has been shown to effectively slow down one-dimensional sliding (Brackley et al, 2013; Gomez and Klumpp, 2016; Li et al, 2009), this effect could be overcome by hopping on DNA.…”

Section: The Target Search Problem: Solving the Speed-stability Pamentioning

confidence: 99%

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins

Kong

Houten

2017

Progress in Biophysics and Molecular Biology

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Since Robert Brown’s first observations of random walks by pollen particles suspended in solution, the concept of diffusion has been subject to countless theoretical and experimental studies in diverse fields from finance and social sciences, to physics and biology. Diffusive transport of macromolecules in cells is intimately linked to essential cellular functions including nutrient uptake, signal transduction, gene expression, as well as DNA replication and repair. Advancement in experimental techniques has allowed precise measurements of these diffusion processes. Mathematical and physical descriptions and computer simulations have been applied to model complicated biological systems in which anomalous diffusion, in addition to simple Brownian motion, was observed. The purpose of this review is to provide an overview of the major physical models of anomalous diffusion and corresponding experimental evidence on the target search problem faced by DNA-binding proteins, with an emphasis on DNA repair proteins and the role of anomalous diffusion in DNA target recognition.

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Molecular crowding enhances facilitated diffusion of two human DNA glycosylases

Cited by 43 publications

References 50 publications

AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

Comparative Effects of Ions, Molecular Crowding, and Bulk DNA on the Damage Search Mechanisms of hOGG1 and hUNG

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins

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