Human alkyladenine DNA glycosylase employs a processive search for DNA damage.
DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that AAG randomly binds to either of the two lesions. AAG-catalyzed base excision creates a repair intermediate, and the subsequent partitioning between dissociation and diffusion to the second site can be quantified from the rates of formation of the different products. Our results demonstrate that AAG has the ability to slide for short distances along DNA at physiological salt concentrations. The processivity of AAG decreases with increasing ionic strength to become fully distributive at high ionic strengths, suggesting that electrostatic interactions between the negatively charged DNA and the positively charged DNA binding surface are important for nonspecific DNA binding. Although the amino terminus of the protein is dispensable for glycosylase activity at a single site, we find that deletion of the 80 amino-terminal amino acids significantly decreases the processivity of AAG. These observations support the idea that diffusion on undamaged DNA contributes to the search for sites of DNA damage.
Hedglin
Mark
M
Chemical Biology Program, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
O'Brien
Patrick J
PJ
D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D055672:Static Electricity; D013379:Substrate Specificity
D011994:Recombinant Proteins; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases