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Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition

Fig 3

Two stages of Rif1 recruitment, their contributions to specificity, and reliance on origin licensing and the replication checkpoint.

Time-lapse confocal microscopy on Rif1-GFP, His2Av-RFP embryos showing the initial binding of Rif1 during transit from one cell cycle to the next. (A) During late anaphase 13, on the approach to cycle 14, chromosomes first exhibit faint and ubiquitous Rif1 staining that accumulates for about 1 min. During the following minute as S phase begins, accumulation continues but is now localized to foci that become clearer as the nuclei swell. See also S2 Movie. (B) During late anaphase 14, on the approach to cycle 15, the early stage of Rif1 staining shows some specificity for the pericentric regions of the chromosomes and forming chromocenter. This specificity is amplified as the interphase 15 nucleus forms and S phase begins. (C) Time-lapse imaging of Rif1-GFP during the normal transition from cycle 13 to 14 and in an embryo injected with purified geminin protein in interphase of 13. Times are indicated with reference to the start of S phase 14. The geminin block to pre-RC formation prevented the recruitment of Rif1 to foci but did not block the initial generalized binding during mitotic exit. (D) Imaging of Rif1-GFP during cell cycle 13 in control (mei41/+) and mei41-null embryos. The Mei41-dependent replication checkpoint is essential during cycle 13 to prevent premature entry into mitosis (10:20 frame). Rif1 foci still form in the absence of a replication checkpoint, but the Rif1 foci are lost earlier, and the premature mitosis leads to bridging and defective cycle 14 nuclei. GFP, green fluorescent protein; His2Av, histone 2A variant; pre-RC, pre-replicative complex; RFP, red fluorescent protein; Rif1, Rap1 interacting factor 1.

Fig 3