Figure 5.

Saw1 recruits Rad1/Rad10 complex to 3′-flap DNA substrate. (A) His-Rad1-Rad10 and His-Saw1 were co-expressed from E. coli and purified together with wild-type His-Saw1, His-saw1-Δ18–24, or His-saw1^DB-, respectively, over a Cobalt column. The 5′-³²P-labelled 3′-flap DNA substrate (0.1 pmol) was then incubated with increasing amounts of the His-Rad1/Rad10/His-Saw1 mixture, reported as μg/ml because the stoichiometry of any complexes is unclear The reaction mixtures were separated by acrylamide mini gel electrophoresis followed by autoradiography to detect Rad1/Rad10/Saw1-DNA or Saw1-DNA complex. Data were assembled by removal of irrelevant lanes and from two gels exposed in parallel. (B) The recruitment of rad1^nuc- to the proximal to the 3′-flap site in EAY1141 and its saw1-R19A or saw1^DB- mutant derivatives was examined by ChIP assays. Fold enrichment is calculated as described in Figure 1. Data represent the mean±s.d. of three or more independent experiments. (C) The top left panel shows a schematic illustration of the SSA substrate and TetO array located ∼15 kb from the HO cut site on chromosome III. The bottom left panel shows representative images of DIC merged with Venus-Rad1 and TetR-mRFP1 either before or after HO induction. Right panel shows percent co-localization of Venus-Rad1 with TetR-mRFP1 in wild-type or isogenic strain deleted for SAW1 and plotted at the indicated time post HO induction. Data represent % co-localization of Venus-Rad1 and TetR-mRFP1 from two independent experiments in each of which scored at least 150 cells and the individual data were plotted vertically together with a line denoting the mean. The size bar represents 0.5 μm.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure S5A ^{(87.8KB, jpg)}