Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism.

The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca(2+) exhibits complex stimulatory and inhibitory effects on the Mg(2+)-dependent catalysis of Ape1, even though Ca(2+) itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.

The human homolog of Escherichia coli Orn degrades small single-stranded RNA and DNA oligomers.

We report here the identification of human homologues to the essential Escherichia coli Orn protein and the related yeast mitochondrial DNA-escape pathway regulatory factor Ynt20. The human proteins appear to arise from alternatively spliced transcripts, and are thus identical, except the human Ynt20 equivalent contains an NH(2)-terminal extension that possesses a predicted mitochondrial protease cleavage signal. In vitro analysis revealed that the smaller human protein exhibits a 3' to 5' exonuclease activity for small (primarily

Mapping the protein-DNA interface and the metal-binding site of the major human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic (AP) endonuclease Ape1 is a key enzyme in the mammalian base excision repair pathway that corrects AP sites in the genome. Ape1 cleaves the phosphodiester bond immediately 5' to AP sites through a hydrolytic reaction involving a divalent metal co-factor. Here, site-directed mutagenesis, chemical footprinting techniques, and molecular dynamics simulations were employed to gain insights into how Ape1 interacts with its metal cation and AP DNA. It was found that Ape1 binds predominantly to the minor groove of AP DNA, and that residues R156 and Y128 contribute to protein-DNA complex stability. Furthermore, the Ape1-AP DNA footprint does not change along its reaction pathway upon active-site coordination of Mg(2+) or in the presence of DNA polymerase beta (polbeta), an interactive protein partner in AP site repair. The DNA region immediately 5' to the abasic residue was determined to be in close proximity to the Ape1 metal-binding site. Experimental evidence is provided that amino acid residues E96, D70, and D308 of Ape1 are involved in metal coordination. Molecular dynamics simulations, starting from the active site of the Ape1 crystal structure, suggest that D70 and E96 bind directly to the metal, while D308 coordinates the cation through the first hydration shell. These studies define the Ape1-AP DNA interface, determine the effect of polbeta on the Ape1-DNA interaction, and reveal new insights into the Ape1 active site and overall protein dynamics.

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 involving Mg2+. We report here that while Ape1 incision of the AP site analog tetrahydrofuran (F-DNA) was approximately 7300-fold reduced in 4 mM EDTA relative to Mg2+, cleavage of ethane (E-DNA) and propane (P-DNA) acyclic abasic site analogs was only 20 and 30-fold lower, respectively, in EDTA compared 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, priming it for enzyme-mediated hydrolysis. Mutating the proposed metal-coordinating residue E96 to A or Q resulted in a approximately 600-fold reduced incision activity for both P and F-DNA in Mg2+compared to wild-type. These mutants, 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 involved in metal coordination were mutated (D70A, D70R, D308A and D308S), but displayed a relatively minor loss of incision activity for F and P-DNA in Mg2+, indicating a non-essential function for these amino acid residues. Mutations at Y171 resulted in a 5000-fold reduced incision activity. A Y171H mutant was fourfold less active than a Y171F mutant, providing evidence that Y171 does not operate as the proton donor in catalysis and that the additional role of E96 may be in establishing the appropriate active site environment via a hydrogen-bonding network involving Y171. D210A and D210N mutant proteins exhibited a approximately 25,000-fold reduced incision activity, indicating a critical role for this residue in the catalytic reaction. A D210H mutant was 15 to 20-fold more active than the mutants D210A or D210N, establishing that D210 likely operates as the leaving group proton donor.

Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases.

Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.