Figure 6.

Novel origin activation is spatially coupled to aberrant replication structures in Chk1^−/− DT40 cells. wt and Chk1-deficient DT40 cells were pulse labelled consecutively with BrdU (20 min) and biotin-dUTP (30 min). Spread DNA fibres were immunolabelled to visualise sites of incorporation of BrdU (green) and biotin-dU (red). Labelling was performed either with or without prior treatment with HCl, as shown (A, B). Merged images show overlays of green and red channels (panels A, B). In panel B, the merged image and individual channels are shown for a high-power view of a single fibre. A diagram describing the analysis is shown (C), using the nomenclature described in Figure 5. X indicates a boundary between regions labelled in the first pulse and a nearby replicon that was labelled predominantly during the second, as seen in panel B (high-power example). Scale bars (5 or 10 μm) are shown on individual images.