Human AP endonuclease 1 stimulates multiple-turnover base excision by alkyladenine DNA glycosylase.
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 multiple-turnover 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 single-turnover 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 in which APE1 displaces AAG from the abasic site, thereby coordinating the first two steps of the base excision repair pathway.
Baldwin
Michael R
MR
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
O'Brien
Patrick J
PJ
D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000502:physiology*; D004795:Enzyme Stability; D006801:Humans; D013379:Substrate Specificity
C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase