Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L

Abstract

Background: Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized.

Methodology/principal findings: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+) and Ca(2+) (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+), Co(2+) and Fe(2+) cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6-7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 μM(-1) · min(-1), respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1.

Conclusions/significance: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.

Figure 1. Protein sequence alignment of putative T. aestivum AP endonuclease (TaApe1L), putative A. thaliana AP endonuclease (AtApe1L), and human APE1.

The deduced amino acid sequences were aligned using ClustalX 2.1. Asterisks (*), colons (:), and periods (.) indicate identical, conservative, and semi-conservative aligned residues, respectively.

Figure 2. Divalent cation dependence of AP endonuclease activity of wheat TaApe1 protein on the oligonucleotide duplex THF•T.

10′-³²P-labelled 30-mer DNA duplex containing a single THF residue was incubated for 5 min at 23°C with 100 nM TaApe1 under BER conditions but in the presence of different divalent cations. Lane 1, 10-mer size marker; lane 2, control non-treated 30-mer duplex THF•T; lane 3, as lane 2 but incubated with 1 nM APE1 for 5 min at 37°C; lane 4, as lane 2 but incubated with 100 nM TaApe1 and 1 mM MgCl₂; lane 5, as lane 4 but in the presence of 10 mM MgCl₂, lane 6, as lane 4 but in the presence of 1 mM CaCl₂ instead of MgCl₂, lane 7, as lane 4 but in the presence of 5 mM CaCl₂ instead of MgCl₂, lane 8, as lane 4 but in the presence of 10 mM CaCl₂ instead of MgCl₂, lane 9, as lane 4 but in the presence of 1 mM MnCl₂ instead of MgCl₂, lane 10, as lane 4 but in the presence of 5 mM MnCl₂ instead of MgCl₂, lane 11, as lane 4 but in the presence of 10 mM MnCl₂ instead of MgCl₂, lane 12, as lane 4 but in the presence of 1 mM CoCl₂ instead of MgCl₂, lane 13, as lane 4 but in the presence of 5 mM CoCl₂ instead of MgCl₂, lane 14, as lane 4 but in the presence of 0.1 mM ZnCl₂ instead of MgCl₂, lane 15, as lane 4 but in the presence of 0.1 mM NiCl₂ instead of MgCl₂, lane 16, as lane 4 but in the presence of 0.1 mM FeCl₂ instead of MgCl₂. For details, see Materials and Methods.

Figure 3. Alignment of human APE1 structure (4LND, the protein backbone and carbons are shown in green) and the homologous model of TaApe1L (the protein backbone and carbons are shown in cyan).

In the amino acid labels, the first label always indicates the nature of the residue and its number in human APE1, the second one, in TaApe1L. The bound metal ion is shown as a magenta van der Waals sphere, the side chains of the protein ligands as sticks. A, metal-binding Site A. Note the difference between Asp in APE1 and Asn in TaApe1L. B, metal-binding site B showing an excellent overlap between the side chain ligands Asp, Asn, and His. The image was prepared using PyMol .

Figure 4. Dependence of the TaApe1L AP endonuclease activity on reaction conditions.

(A) pH dependence, (B) concentration of Mn²⁺, (C) ionic strength, and (D) incubation temperature. For details see Materials and Methods.

Figure 5. Time-dependent cleavage of the oligonucleotide duplex containing a synthetic AP site by TaApe1L.

10′-³²P-labelled 30-mer TH•T was incubated with 5 nM TaApe1L at room temperature. (A) Separation of the reaction products by denaturing PAGE. (B) Graphical representation of time course of the TaApe1L-catalyzed cleavage of THF•T. For details, see Materials and Methods.

Figure 6. DNA repair activities of TaApe1L on gapped oligonucleotide duplexes containing 3′-P, 3′-PA and 3′-OH termini.

10′-³²P-labelled 34-mer U•G duplex was treated first with hUNG/Fpg, hUNG/Nth or hUNG/Nfo and then incubated with either 5 nM APE1 at 37°C or with TaApe1L at room temperature under the respective optimal reaction conditions. For details, see Materials and Methods.

Figure 7. Presence of the TaApe1L protein in wheat seedling tissues.

Wheat seedlings were grown for 4 days and then dissected into shoot, root, aleurone and scutellum tissues. 35 μg of soluble protein extracted from each tissue were separated using 10% SDS-PAGE, transferred to the membrane, and incubated with rabbit anti-TaApe1L polyclonal antiserum. Lane 1, 10 ng of the recombinant TaApe1L protein; lane 2, 5 ng of TaApe1L; protein extracts from shoot (lane 3), root (lane 4), scutellum (lane 5), and aleurone (lane 6). For details, see Materials and Methods.