Phospho-site mutant Rif1 does not dissociate from chromatin and blocks replication.

<p>(A) Schematic (above) of Rif1 protein with N-terminal HEAT repeats (orange), putative DNA-binding domain (green), and PP1 interaction motifs (purple boxes). The amino acid sequence (below) of the indicated portion of the Rif1 protein shows candidate CDK and DDK phosphorylation sites (red). These 15 S/T residues were mutated to alanine to create the Rif1.15a phospho-mutant allele. (B) Left panel shows anti-gfp WBs used to detect Rif1-GFP from 1-h-old embryos. Protein extracts were treated with lambda phosphatase or buffer only for 1 h and then were run on SDS-PAGE gels cast with phos-tag to decrease the migration of phosphorylated proteins. Right panel shows phos-tag WBs used to detect Rif1-GFP from embryos aged to 1 h (pre-MBT) or to 2.5 h (MBT). Asterisk denotes background band. (C) Ectopic expression of the indicated versions of Rif1 by injection of mRNA during cycle 11. After injection, embryos were filmed during mitosis 13. The yellow arrow points to an example of an anaphase bridge. (D) Images of flies (left) and a pharate pupa (right) show the consequence of expression of either Rif1 (middle) or Rif1.15a (right) in the developing fly eye/head capsule using Eyeless-Gal4. (E) Anti-GFP immunoprecipitation followed by western blotting with the indicated antibodies. Protein extracts were prepared from embryos harvested before the MBT (1 h old) or after aging to the MBT (2.5 h old). Phos-Y15 (left-bottom) indicates the inactive phosphorylated form of Cdk1. (F) Frames from live imaging of nuclei starting in G2 of 14 and progressing into a defective mitosis following expression of Rif1.15a-GFP from injected mRNA. Yellow arrows indicate extensive anaphase bridging with a residue of Rif1.15a-GFP. Magnified images (right) show progression of one nucleus. CDK, cyclin-dependent kinase; DDK, Dbf4-dependent kinase; GFP, green fluorescent protein; His2Av, histone 2A variant; IP, immunoprecipitation; MBT, mid-blastula transition; PP1, protein phosphate 1; RFP, red fluorescent protein; Rif1, Rap1 interacting factor 1; UAS, upstream activating sequence; WB, western blot; WT, wild type.