File S1 - Cloning and Characterization of a Wheat Homologue of Apurinic/Apyrimidinic Endonuclease Ape1L

Figures S1–S6. Figure S1. SDS-PAGE analysis of the purified recombinant TaApe1 protein. Lane 1, protein size markers; lane 2, human APE1, 2 μg; lane 3, TaApe1, 1 μg; lane 4, TaApe1, 5 μg. Figure S2. Protein sequence alignment of human APE1, Arabidopsis thaliana AP endonucleases AtApe1L, AtArp and AtApe2. 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 S3. Divalent cation dependence of wheat TaApe1-catalyzed activities on the oligonucleotide duplex THF•T. 10 nM 5′-32P-labelled 30-mer DNA duplex containing a single THF residue was incubated for 10 min at 23°C with 5 or 10 nM TaApe1 under standard reaction conditions but in the presence of different divalent cations. (A) Effects of MgCl2, CaCl2 and CoCl2 on the enzyme activities (B) Effects of ZnCl2, NiCl2 and FeCl2 on the enzyme activities. Lanes 1, 3, 7, 11, control 30-mer duplex THF•T incubated in reaction buffer without enzyme; lane 2, as lane 1 but incubated with 1 nM APE1 for 5 min at 37°C; lanes 4–6, 8–10 and 12–14, 30-mer duplex THF•T incubated with TaApe1L in the presence of the indicated metal chloride. For details, see Materials and Methods. Figure S4. Effects of the absence of divalent cations and/or presence of 1 mM EDTA in the reaction buffer on the TaApe1L AP endonuclease activity. 10 nM 5′-32P-labelled 30-mer DNA duplex containing a single THF residue was incubated with 5 nM TaApe1L at 23°C. Lane 1, control non-treated THF•T duplex; lane 2, as lane 1 but incubated for 5 min at 37°C with 1 nM APE1; lane 3, as lane 2 but incubated with 5 nM TaApe1L and 1 mM MnCl2 for 5 min at 23°C; lane 4, as lane 3 but incubated with 10 nM TaApe1L: lane 5, as lane 3 but incubated with 0 mM MnCl2; lane 6, as lane 5 but incubated with 10 nM TaApe1L; lane 7, as lane 3 but incubated with 1 mM EDTA; lane 8, as lane 7 but incubated with 10 nM TaApe1; lane 9, as lane 3 but incubated with 5 mM EDTA; lane 10, as lane 9 but incubated with 10 nM TaApe1. The arrows mark the position of the 30-mer DNA substrate and 10-mer cleavage product. For details, see Materials and Methods. Figure S5. Preparation of the DNA substrates containing a nick flanked with 3′-PA or 3′-OH termini or a 1-nt gap flanked with 3′-P to measure 3′-repair phoshodiesterase activities of TaApe1L. (A) Schematic representation of the structures of 3′ and 5′ DNA termini at a DNA nick or gap. (B) Separation of 5′-32P-labelled oligonucleotide fragments by denaturing PAGE. 10 nM 5′-32P-labelled 34-mer DNA duplex containing a single U•G base pair was incubated for 5 min at 37°C with 10 nM hUNG and then with either 20 nM Fpg or 20 nM Nth or 20 nM Nfo for 15 min at 37°C. Lane 1, control non-treated 34-mer duplex U•G; lane 2, as lane 1 but incubated with 10 nM hUNG and 20 nM Fpg; lane 3, as lane 1 but incubated with 10 nM hUNG and 20 nM Nth; lane 4, as lane 1 but incubated with 10 nM hUNG and 20 nM Nfo. The arrows mark the position of the full-length 34-mer substrate and 19-mer cleavage fragments containing by 3′-PA, 3′-OH and 3′-P. For details, see Materials and Methods. Figure S6. Activities of human APE1 and TaApe1L on αdA•T and DHU•G oligonucleotide duplexes. 10 nM 5′-32P-labelled 30-mer DNA duplex containing a single modified residue was incubated with 5 nM TaApe1L at 23°C. Lane 1, control non-treated THF•T duplex; lane 2, as lane 1 but treated with 1 nM APE1 for 5 min at 37°C; lane 3, as lane 1 but treated with 5 nM TaApe1L for 5 min; lane 4, as lane 3 but treated for 10 min; lane 5, as lane 3 but treated for 15 min; lane 6, as lane 3 but treated for 30 min; lane 7, control non-treated αdA•T duplex; lane 8, as lane 7 but treated with 5 nM APE1 for 5 min at 37 °C; lane 9, as lane 7 but treated with 5 nM TaApe1L for 5 min; lane 10, as lane 9 but treated for 10 min, lane 11, as lane 9 but treated for 15 min, lane 12, as lane 9 but treated for 30 min; lane 13, control non-treated DHU•G duplex; lane 14, as lane 13 but treated with 5 nM APE1 for 5 min at 37°C; lane 15, as lane 13 but treated with 5 nM TaApe1L for treated for 5 min; lane 16, as lane 15 but treated for 10 min, lane 17, as lane 15 but treated for 15 min, lane 18, as lane 15 but treated for 30 min. The arrows mark the position of the full-length 30-mer substrate and 10-mer AP-endonuclease and NIR cleavage products. For details, see Materials and Methods. (PDF)