Specificity and Searching Mechanism of Alkyladenine DNA Glycosylase.

Abstract

Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway, and it is responsible for locating and excising alkylated and deaminated base lesions such as 1,N6-ethenoadenine (εA) and hypoxanthine (Hx). AAG uses facilitated diffusion to search for target lesion sites via nonspecific electrostatic interactions. Previous studies have shown that AAG is capable of hopping over tightly bound roadblock proteins on DNA. This enables AAG to perform redundant searching on short DNA segments, but it has the pitfall of potentially trapping the enzyme in local regions. We show that AAG uses intersegmental transfer to balance local and global searching. Intersegmental transfer is more prominent at high DNA concentrations, suggesting an important contribution of this searching mode in the crowded nucleus. We next investigated excision efficiency of AAG on the εA substrate using pulse-chase assays to separate searching and catalysis. Results show that the commitment of AAG for εA excision is salt dependent, and increasing salt concentration results in decreased excision efficiency. Furthermore, excision efficiency was found to be dependent on the chase concentration at low salt, which is consistent with the aforementioned intersegmental transfer model at high chase DNA concentrations. We then compared catalytic specificity of AAG on εA opposing different bases and on εA•T and Hx•T lesions. At low salt, all substrates tested had similar kcat/KM values due to facilitated diffusion. At high salt, AAG excises εA opposing different bases with different kcat/KM: εA•C ≈ εA•T > εA•A > εA•G. It was also shown that the εA•T lesion is a preferred substrate over the Hx•T lesion by a factor of over 100-fold. Finally, we tested how the in vitro kinetic parameters of AAG are related to its in vivo function. Eight single point mutants at the DNA-AAG binding interface were tested, and we showed that kcat/KM and searching efficiency are correlated with each other, and also show a rough correlation with the in vivo function of AAG. This provides evidence that the search for DNA damage can limit the rate of repair in cells. Taken together, these studies shed light on the searching and catalytic mechanisms of AAG.