DNA-binding and double-strand break formation by Vpr.

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DNA-binding and double-strand break formation by Vpr. DNA-binding and double-strand break formation by Vpr. A, purification of VprΔC12. Purified VprΔC12 was analyzed using 16% SDS-PAGE with Coomassie brilliant blue staining. Lane 1, molecular mass markers. Lanes 2 and 3, purified wild-type Vpr and VprΔC12 protein, respectively. B, the DNA-binding activity of VprΔC12. The DNA-binding experiments were done using the protocol used to obtain Fig. 2B. The concentrations of VprΔC12 were 2.5 μmol/L (lanes 2 and 9), 5 μmol/L (lanes 3 and 10), and 10 μmol/L (lanes 4 and 11), and those of the wild-type Vpr were 2.5 μmol/L (lanes 5 and 12), 5 μmol/L (lanes 6 and 13), and 10 μmol/L (lanes 7 and 14). Negative controls without protein (lanes 1 and 8) are included. C, PFGE analysis of double-strand breaks in isolated nuclei treated with Vpr or VprΔC12. Molecular mass marker (lane 1), cells without (lane 2) or with (lane 3) 7.5 Gy of X-ray irradiation, control nuclei (lane 4), nuclei with Vpr (lane 5), and nuclei with VprΔC12 (lane 6). Vpr was used at 10 μmol/L. Arrow, double-strand breaks. Hiroaki Tachiwana et al. Cancer Res 2006;66:627-631 ©2006 by American Association for Cancer Research