Rif1 dissociates from chromatin before the underlying sequences replicate.

<p>(A) Stills from time-lapse microscopy of Rif1-GFP and mCherry-PCNA during S phase 14 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005687#pbio.2005687.s007" target="_blank">S3 Movie</a>). Rif1 marks numerous satellites early, and the number of foci progressively declines. After early widespread staining, PCNA, a marker of active replication, transiently marks a progression of different satellites. Though both Rif1-GFP and mCherry-PCNA form foci on satellite sequences, the signals do not overlap. At different positions, the period of PCNA recruitment follows loss of Rif1. For example, the yellow arrows track a Rif1 focus as it disappears. In the paired mCherry-PCNA images, recruitment occurs just as Rif1 disappears. Note that the mCherry-PCNA signal is much expanded, in agreement with previous findings that PCNA recruitment is coincident with decompaction of satellite foci [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005687#pbio.2005687.ref026" target="_blank">26</a>]. Below, a single z plane from 10 min into S phase 14 shows that even at this early stage of S phase when mCherry-PCNA is widespread, there are “holes” in its distribution, and the signal from Rif1 falls within these holes. (B) Stills from time-lapse microscopy of Rif1-GFP and mCherry-PCNA during S phase 15 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005687#pbio.2005687.s009" target="_blank">S5 Movie</a>). During this S phase, the late-replicating heterochromatin is clustered into an obvious chromocenter. Within this cluster, replicating and nonreplicating satellites are nearby, but each satellite forms a coherent focus and replicates at different times [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005687#pbio.2005687.ref026" target="_blank">26</a>]. As in S phase 14, Rif1 marks late-replicating chromatin, and Rif1 dissociates from these sequences before the acquisition of PCNA. Yellow arrows track the dissociation of Rif1 from one focus, followed by its recruitment of mCherry-PCNA as it decompacts and replicates. Below, a single z plane from 52 min into S phase 15 shows that Rif1 does not colocalize with PCNA. The outline of Rif1 foci is traced in yellow on the still image of PCNA. Note that the single-plane images do not show all of the nuclei seen in the merged stack shown above in (B). (C) Live imaging of Rif1 and the satellite 1.686 using an mCherry-tagged TALE protein (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005687#pbio.2005687.s010" target="_blank">S6 Movie</a>). Yellow arrow indicates the decompaction and replication of 1.686. After its replication, the 1.686 repeat recompacts and appears brighter. Rif1 dissociates from this sequence prior to its replication. (D) Nuclei in S phase 14 stained for Rif1 and for the Y-chromosomal satellite repeat AATAC. Rif1 is bound to AATAC during early S14 before the AATAC repeat replicates. During late S14, the duplicated AATAC sequence lacks Rif1 stain. EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; PCNA, proliferating cell nuclear antigen; Rif1, Rap1 interacting factor 1; TALE, transcription activator-like effector.