Figure 8: HOP2-MND1 induces conformational changes upon interaction with RAD51.

(a) His-hRAD51 (5 μM) was digested by trypsin (19 μg ml⁻¹) in the absence (lanes 2–5) or presence of mHOP2-MND1 (0.5 μM; lanes 6–9). Where indicated the reaction contained ϕX174 ssDNA (15 μM, nucleotides) (lanes 3, 5, 7 and 9) and/or ATP (1 mM) (lanes 4, 5, 8 and 9). The hRAD51 fragments were resolved in a 12% NuPAGE Bis-Tris gel (Life Technologies) in MES buffer and visualized by western blotting using anti-HIS tag monoclonal antibody (Abgent). Numbers on the left indicate approximate size of the fragments (kDa) as determined by positions of His-tagged BenchMark markers. The region of the gel corresponding to a particular part of hRAD51 undergoing conformational changes upon addition of DNA or/and nucleotide cofactors is highlighted as enhanced in the inset. The fragments relevant to the discussion in the text are marked by roman numbers I, II and III. Untreated hRAD51 (38 kDa) is shown in lane 1. His-tagged BenchMark markers (Invitrogen) are denoted by ‘M’. (b) The relative intensity of each fragment shown in the inset is plotted as a fraction of the sum of intensities of fragments I–III. The digestion patterns were reproduced in four (for the trypsin digestion in the absence of mHOP2-MND1) or three (digestion in the presence of the HOP2–MND1 complex) independent experiments. Error bars represent the s.d.