Figure 2.

Assay of in vitro DNA binding by full-length DBP1 and AtDBP1. Recombinant full-length DBP proteins were expressed in E. coli bearing a C-terminal hexa-His tag, purified by nickel affinity chromatography according to the manufacturer's instructions (Qiagen, Valencia, CA), and assayed for their ability to bind to the double-stranded 3a4 oligonucleotide probe as described (Carrasco et al., 2003). Briefly, complementary oligonucleotides leaving 5′ protruding ends were annealed and radioactively labeled using the Klenow fragment of E. coli DNA polymerase I. The sequences of the oligonucleotides used to build the double-stranded 3a4 probe are the following, with the nucleotides modified in the mutated 3a4mut probe shown in bold: 3a4-plus, 5′-TCGACTAGGCGGCTAATATTTGCCTTTGTCTCCCTC-3′; and 3a4-minus, 5′-TCGAGAGGGAGACAAAGGCAAATATTAGCCGCCTAG-3′. Purified proteins (1 μm) were incubated for 10 min at room temperature with the radiolabeled DNA probe (0.3 nm) in binding buffer [20 mm HEPES-KOH, pH 7.6, 4 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol, 10% (v/v) glycerol], prior to separation on a native 6% (w/v) polyacrylamide gel in 0.5× Tris-borate/EDTA buffer.