FIGURE 7.

DNA translocase and nucleosome sliding activity of GG-NER complex. A, triple helix substrates consisting of a 40-nucleotide triple helical region were prepared as described under “Experimental Procedures.” Substrates were incubated with (lanes 6–7) or without (lane 3) GG-NER complex at 30 °C for 30 min in the presence of ATP (lanes 5 and 7) or ATPγS (lane 6) or heated for 5 min at 90 °C (lane 2). SV-40 T-antigen-treated sample (lane 1) is included as a positive control. Lane 4 contains [γ-³²P]dATP-labeled third strand (indicated as Free TFO) only. A control fraction from the final step of the purification known not to contain GG-NER complex was also tested (lane 5). Reaction mixtures were separated on a 1% agarose gel, and displacement of the free triplex-forming oligonucleotide (TFO) was monitored. B, movement of nucleosomes on the Cy3-labeled DNA fragment 105A64 was monitored following heating at 47 °C for 1 h (lane 2) or treatment with different GG-NER complex-containing fractions from the final step of the purification as indicated previously (16) (lanes 4–11) or 0.12 pmol of RSC complex (lane 3) for 30 min by native gel electrophoresis.