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. 2006 May 10;34(9):2508-15.
doi: 10.1093/nar/gkl259. Print 2006.

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs

Peter Burkovics  1 , Valeria Szukacsov, Ildiko Unk, Lajos Haracska
Affiliations

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs

Peter Burkovics et al. Nucleic Acids Res. .

Abstract

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.

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Figures

Figure 1
Figure 1
Purification of the wild type and mutant human Ape2. (A) Schematic representation of human Ape2. The N-terminus of Ape2 contains the conserved ExoIII domain characteristic of class II AP endonucleases, while the C-terminus is unique to Ape2-subfamily. The arrow indicates the amino acid D227 residue that was changed to alanine in the active site of Ape2. (B) Purity of wild type and mutant Ape2 proteins. Purified wild-type GST–Ape2 and GST–Ape2D277A fusion proteins were analyzed on a 10% denaturating polyacrylamide gel and stained with Coomassie blue. Lane 1, molecular weight standard; lane 2, 0.5 μg of purified GST–Ape2; lane 3, 0.5 μg of purified GST–Ape2D277A. The arrow identifies full-length GST–Ape2, while asterisk indicate the co-purifying proteolytic product of Ape2.
Figure 2
Figure 2
AP-endonuclease activity of Ape2. (A) The AP-endonuclease activity of Ape2 was assayed using a 75 nt double-stranded DNA substrate (S1) containing a single AP-site at position 31 on the 5′-labeled strand. The DNA substrate (10 nM) was incubated without (lane 1) or with wild type (lanes 2–3) or D277A mutant Ape2 (lanes 4–5) in standard reaction buffer but containing 150 mM NaCl for 5 min at 37°C. The reaction products were analyzed on 10% polyacrylamide gels containing 8 M urea, and the DNA bands were visualized by autoradiography. The position of the incision product at 30 nt is indicated. (B) Comparison the specificity of Ape2 and Ape1 endonuclease activities. The DNA substrate (S1) was incubated without (lane 1) or with Ape1 (lanes 2 and 4) or Ape2 (lanes 3 and 5) in the presence or absence of 150 mM NaCl in standard reaction buffer for 5 min at 37°C. (C) The NaCl concentration dependence of the endonuclease activity of Ape2. S1 DNA substrate (10 nM) was incubated with Ape2 (25 nM) in standard reaction buffer supplemented with 0–300 mM NaCl as indicated.
Figure 3
Figure 3
3′-Exonuclease and 3′-phosphodiesterase activities of Ape2. (A) The 3′–5′ exonuclease activity of wild-type (lanes 2 and 3) and mutant (lanes 4 and 5) Ape2 was assayed on partial DNA duplex (S3) (10 nM) in standard reaction buffer for 5 min at 37°C. (B) The phosphodiesterase activity of Ape2 was tested on a DNA substrate (S2) containing a 5′-labeled 35 nt oligomer with a 3′-PG terminus annealed to a 70 nt oligomer. Wild type (lanes 2 and 3) and mutant (lanes 4 and 5) Ape2 proteins were incubated with 10 nM DNA substrates in standard reaction buffer for 5 min at 37°C. The positions of the reaction product and the substrate are indicated by 3′-OH and 3′-PG, respectively.
Figure 4
Figure 4
Optimal reaction conditions for the 3′–5′ exonuclease activity of the Ape2. The 3′–5′ exonuclease activity of GST–Ape2 was assayed under various reaction conditions using a partial DNA duplex (S3) (10 nM) in which the 5′-labeled oligonucleotide contained a 3′-recessed terminus. (A) Metal ion dependence. The first lane contains reaction mixture without any metal ions in the reaction buffer. Reactions were carried in the presence of 8 mM MgCl2 (lanes 2–4) or 0.5 mM MnCl2 (lanes 5–7) and increasing concentrations of Ape2 as indicated. (B) Graphical representation of results in (A); (C) NaCl concentration dependence. The first lane contains reaction without any Ape2 protein. (D) Graphical representation of results in (C); (E) pH dependence. (F) Graphical representation of results in (E).
Figure 5
Figure 5
Exonuclease activity of Ape2 protein on different DNA substrates. (A) In each reaction single-stranded (lanes 1 and 2), 3′ overhanging partial heteroduplex (lanes 3 and 4), blunt-ended (lanes 5 and 6), 3′ recessed partial heteroduplex (lanes 7 and 8), or single nucleotide gap containing heteroduplex DNA substrates (lanes 9 and 10) (10 nM) were incubated with Ape2 (5 nM) for 10 min at 37°C. (B) Exonuclease activity of Ape2 on nick or gap containing DNA duplexes. Parallel reactions were carried out using 5 nM of Ape2 and 10 nM DNA duplex containing a nick, or 1, 2, 3 or 4 nt gap, or a 3′ recessed partial heteroduplex DNA as indicated. Asterisks indicate the radioactively labeled terminus. (C) Graphical representation of results in (B).
Figure 6
Figure 6
Comparing the 3′ exonuclease activity of Ape2 on paired and mispaired DNA substrates. The 3′–5′ exonuclease activity of GST–Ape2 (5 nM) was assayed on partial DNA duplexes (10 nM) containing all the possible 16 matched and mismatched nucleotide pairs at the primer–template junctions; control reactions without Ape2 (lanes 1, 6, 11 and 16); exonuclease reactions on the correctly paired 3′ end substrates (lanes 3, 7, 15 and 19) and on mispaired DNA (lanes 2, 4, 5, 8–10, 12–14, 17, 18 and 20). The products of three independent experiments, of which one is shown on top, were quantified by PhosphorImager, and the cleavage efficiency of the Ape2 exonuclease on these various DNA substrates are shown under each lane by diagrams.
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