Redox state-dependent modulation of DNA bending and end-joining by HMGB1.

<p>(<b>A</b>) DNA bending by HMGB1. ³²P-labeled 123-bp DNA duplex (∼0.2 nM) was pre-incubated with different amounts of reduced or oxidized HMGB1 (6, 10, 15, 35, 75, 150 and 300 nM; lanes 2–8), followed by ligation with T4 DNA ligase as detailed under “<i>Materials and Methods</i>”. (<b>B</b>) Percentage of DNA minicircles from ligase-mediated circularization assay in the presence of reduced or oxidized HMGB1. 100% denotes production of DNA minicircles at 75 nM reduced HMGB1 protein. Presented data are based on four independent experiments. redHMGB1, reduced HMGB1; oxHMGB1, oxidized HMGB1. (<b>C</b>) DNA bending by HMGB1. ³²P-labeled 66-bp DNA duplex (∼1 nM) was pre-incubated with different amounts of reduced or oxidized HMGB1 (50, 100, 150, 200, 250 and 400 nM; lanes 3–8), followed by ligation with T4 DNA ligase as detailed under “Materials and Methods”. Lane 1, no ligase added. (<b>D</b>) DNA end-joining by HMGB1. The ³²P-labeled 123-bp DNA fragment (∼2 nM) was pre-incubated with different amounts of reduced or oxidized HMGB1 (10, 20, 40, 60, 100, 150 and 200 nM; lanes 2–8), followed by ligation with T4 DNA ligase as in panels (<b>A</b>) and (<b>B</b>). Lane 16 in panel (<b>D</b>) corresponds to the reaction in lane 14 in which the oxidized HMGB1 was pre-treated with 10 mM DTT before addition of ligase. L, linear 123-bp DNA duplex.