title: AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product.
abstract: 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, could be detected 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 had been deleted. Stimulation by APE1 does not involve relief from product inhibition because thymine DNA glycosylase, an enzyme that binds more tightly to AP sites than hOGG1 does, 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 cross-linking assays. The combined results indicate a mechanism whereby 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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Esadze
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
authors.forename: Gaddiel
authors.initials: G
authors.lastname: Rodriguez
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
authors.forename: Shannen L
authors.initials: SL
authors.lastname: Cravens
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
authors.forename: James T
authors.identifier: 0000-0003-2572-7807
authors.initials: JT
authors.lastname: Stivers
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005976:Glutaral / Q000737:chemistry; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
chemical_list: D003432:Cross-Linking Reagents; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D000255:Adenosine Triphosphate; D004247:DNA; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D005976:Glutaral