Figure 9.

Excision of a cisplatin-1,2-intrastrand d(GpG) cross-link by active repair extracts in the presence of HMGB1 or HMGB4. (A) Platinated or unplatinated 146-bp repair substrates were incubated in the presence of added HMGB1 or HMGB4 30 °C. The samples were collected after 1 h or 3 h. The repair products were resolved by 8% urea-PAGE. In control experiments, no excision products were observed when an unplatinated probe was incubated with repair active cell free extract (lane 1), whereas they were observed when a platinated substrate was incubated with cell extracts in the absence of recombinant HMGB proteins (lane 2). Concentrations of HMGB proteins were increased, from 125 nM (lane 3 and lane 10) to 8 µM (lane 9 and lane 16). (B) The excision products from a platinated repair substrate from 1 h (left) or 3 h (right) incubation. Extended incubation periods caused a noticeable increase of minor, lower molecular weight, products, that might be the consequence of exonucleolytic degradation of primary repair products. (C) Plot of the fraction of repaired substrate versus amount of added HMGB1 or HMGB4 protein after 1 h and 3 h incubation. The repair fraction, obtained by integrating excision products in the ~20–30 bp region of a given lane in the gel and dividing that number by the total intensity in that lane, was normalized to the fraction of repair product in a reaction lacking any HMGB protein (lane 2 of A). Longer incubation times increased the total amounts of repair products, but the concentration-dependent repair inhibition ability of HMGB proteins was not influenced by extending the incubation time.