Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of damaged purine bases from DNA, including hypoxanthine that is formed by the oxidative deamination of adenine. We used steady state, pre-steady state, and single-turnover kinetic assays to show that the multipleturnover excision of hypoxanthine in vitro is limited by release of the abasic DNA product. This suggests the possibility that the product release step is regulated in vivo by interactions with other base excision repair (BER) proteins. Such coordination of BER activities would protect the abasic DNA repair intermediate and ensure its correct processing. AP endonuclease 1 (APE1) is the predominant enzyme for processing abasic DNA sites in human cells. Therefore, we have investigated the functional effects of added APE1 on the base excision activity of AAG. We find that APE1 stimulates the multiple-turnover excision of hypoxanthine by AAG, but has no effect on singleturnover excision. Since the amino terminus of AAG has been implicated in other protein-protein interactions we also characterize the deletion mutant lacking the first 79 amino acids. We find that APE1 fully stimulates the multiple-turnover glycosylase activity of this mutant, demonstrating that the amino terminus of AAG is not strictly required for this functional interaction. These results are consistent with a model whereby APE1 displaces AAG from the abasic site, thereby coordinating the first two steps of the base excision repair pathway.Single base lesions are the most frequently occurring type of DNA damage and the majority of these are repaired by the base excision repair (BER) 1 pathway (1). The BER pathway is initiated by a DNA repair glycosylase that is responsible for finding the base lesions, flipping out the damaged nucleotide into the active site, and catalyzing hydrolysis of the N-glycosidic bond. Cells contain a diverse repertoire of DNA repair glycosylases that collectively are able to recognize hundreds of distinct base lesions (2-4). In the case of monofunctional glycosylases the products are the nucleobase lesion and an apurinic/apyrimidinic (AP) DNA duplex. Subsequently, AP endonuclease 1 (APE1) hydrolyzes the phosphodiester bond immediately 3 to the AP site to create a single strand break with a 3´-hydroxyl and a 5´-deoxyribose † This work was supported by a grant from the NIH to P.O. (CA122254).
SUPPORTING INFORMATION AVAILABLEPre-incubation controls establishing the stability of Δ80 AAG, formamide quenched controls to show that APE1 has little residual activity in the absence of magnesium ion, linear concentration dependence of burst amplitude and steady state rate, demonstration that the singleturnover reaction is saturated at low ionic strength, and an additional experiment showing that one equivalent of product abolishes the burst phase. This material is available free of charge via the internet at http://pub.acs.org. 1 Abbreviations: AAG, Alkyladenine DNA glycosylase, also known as methylpurine DNA glycosylase (MPG) and 3-methylad...