TY - JOUR
T1 - The role of Mg2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease
T2 - New insights from EDTA-resistant incision of acyclic abasic site analogs and site-directed mutagenesis
AU - Erzberger, Jan P.
AU - Wilson, David M.
N1 - Funding Information:
We thank Ms Tina Xi and Dr Matthew Coleman for invaluable sequencing support, Drs Felice Lightstone, Daniel Barsky, Michael Colvin and members of the Wilson lab for helpful discussions and Dr James George for critical input. Drs Masaru Takeshita and Arthur Grollman kindly provided the main E and primary P-DNA stocks. This work was carried out under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under contract no. W-7405-ENG-48 and supported by LDRD (97-ERD-002) and NIH (CA79056) grants to D.M.W.
PY - 1999/7/9
Y1 - 1999/7/9
N2 - Apel, 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 Apel incision of the AP site analog tetrahydrofuran (F-DNA) was ~ 7300-fold reduced in 4 mM EDTA relative to Mg2+, cleavage of ethane (E-DNA) and proyane (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 ~ 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 ~ 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.
AB - Apel, 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 Apel incision of the AP site analog tetrahydrofuran (F-DNA) was ~ 7300-fold reduced in 4 mM EDTA relative to Mg2+, cleavage of ethane (E-DNA) and proyane (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 ~ 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 ~ 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.
KW - Ape1
KW - DNA repair
KW - Hap1
KW - Nuclease
KW - Phosphate hydrolysis
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U2 - 10.1006/jmbi.1999.2888
DO - 10.1006/jmbi.1999.2888
M3 - Article
C2 - 10390343
AN - SCOPUS:0033538343
SN - 0022-2836
VL - 290
SP - 447
EP - 457
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -