Fig. 4. Purified recombinant MRE11 from Thermotoga maritima removes topoisomerase IIα covalent complexes from genomic DNA.

Purified recombinant MRE11 from Thermotoga maritima (A) SDS-PAGE of TmMre11, TmMre11H94S, TmMre11H61S or TmMre11H180S proteins. (B) Endonuclease assays were performed with single-stranded bacteriophage φX174 DNA as a substrate with TmMre11, TmMre11H94S, TmMre11H61S or TmMre11H180S. K562 cells were treated with 100 µM etoposide for two hours prior to being embedded in agarose on microscope slides. Slides bearing agarose-embedded cells (1–2×10⁶ cells in 100 µl 1% agarose in PBS spread across the slide surface) were incubated with MRE11 buffer (positive control) or 1 µg TmMre11, TmMre11H94S, TmMre11H61S or TmMre11H180S. Quantitative immunofluorescence was carried out for topoisomerase IIα (C) or -β (D). The mean fluorescence for each nucleus was normalised to the positive control, and the mean±SEM are shown. ***  =  p value, 0.0001.