Distribution of ORCs and GINS on extended chromatin fibers with longer EdU labeling times. (A to D) ORC1 (red signal), Psf1 (blue signal), EdU tracks (green signal); (E) ORC2 (red signal), EdU tracks (green). (A) Chromatin fiber from cells labeled with EdU for 30 min. (B to E) Chromatin fibers from cells that were labeled for 40 min with EdU. As replication proceeds, the signals from ORCs and Psf1 are no longer visible on chromatin fibers. We also demonstrate here that the depletion of ORC and GINS complex proteins from some fibers is specific and not the result of uneven antibody staining or differences in microenvironments on the slide. This specific depletion can be seen in Panel B, where there are unreplicated regions that still have associated ORC1 and Psf1 proteins (arrow heads) on the same fiber with regions that have completed replication and are devoid of these proteins. Also, in Panel D we show two adjacent fibers, one fiber with immunofluorescent signal from ORC1 and Psf1 but with no EdU staining, the other fiber with only EdU staining and no ORC1 and Psf1 antibody staining. Bars ≅ 25 μm (≅ 400 kb).