The role of Mg2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease: New insights from EDTA-resistant incision of acyclic abasic site analogs and site-directed mutagenesis

Ape1, the major protein responsible for excising apurinic/apyrimidinic (AP) sites from DNA, cleaves 5' to natural AP sites via a hydrolytic reaction i nvolving Mg2+. We report here that while Ape1 incision of the AP site analo g tetrahydrofuran (F-DNA) was similar to 7300-fold reduced in 4 mM EDTA rel ative to Mg2+, cleavage of ethane (E-DNA) and propane (P-DNA) acyclic abasi c site analogs was only 20 and 30-fold lower, respectively, in EDTA compare d to Mg2+. This finding suggests that the primary role of the metal ion is to promote a conformational change in the ring-containing abasic DNA, primi ng it for enzyme-mediated hydrolysis. Mutating the proposed metal-coordinat ing residue E96 to A or Q resulted in a similar to 600-fold reduced incisio n activity for both P and F-DNA in Mg2+ compared to wild-type. These mutant s, while retaining full binding activity for acyclic P-DNA, were unable to incise this substrate in EDTA, pointing to an alternative or an additional function for E96 besides Mg2+-coordination. Other residues proposed to be i nvolved in metal coordination were mutated (D70A, D70R, D308A and D308S), b ut displayed a relatively minor loss of incision activity for F and P-DNA i n Mg2+, indicating a non-essential function for these amino acid residues. Mutations at Y171 resulted in a 5000-fold reduced incision activity. A Y171 H mutant was fourfold less active than a Y171F mutant, providing evidence t hat Y171 does not operate as the proton donor in catalysis and that the add itional role of E96 may be in establishing the appropriate active site envi ronment via a hydrogen-bonding network involving Y171. D210A and D210N muta nt proteins exhibited a similar to 25,000-fold reduced incision activity, i ndicating a critical role for this residue in the catalytic reaction. A D21 0H mutant was 15 to 20-fold more active than the mutants D210A or D210N, es tablishing that D210 likely operates as the leaving group proton donor. (C) 1999 Academic Press.