Identification of the critical replication targets of CDK reveals direct regulation of replication initiation factors by the embryo polarity machinery in C. elegans
Fig 2
Two CDK sites in SLD-3 are essential for the interaction with MUS-101 A.
Alignment of the CDK sites in Sld3/Treslin required for the interactions with Dpb11/TopBP1. The amino acid numbers of the two orthologous CDK sites in C. elegans SLD-3 are indicated below. B. Western blot of Gal4-SLD-3 (389–557) wild type (WT) or the 2A mutant expressed in yeast arrested either in G1 phase (low CDK activity) with alpha factor, or arrested in G2 phase (high CDK activity) with nocodazole. C. Yeast two-hybrid analysis between MUS-101 (1–448) and SLD-3 (389–557) wild type (WT) or with the two CDK sites threonine 438 and 487 mutated to alanine (2A), aspartic acid (2D) or glutamic acid (2E). D and E. Box and whisker plot of embryonic lethality after sld-3 RNAi by injection as in Fig 1C. The extra, RNAi insensitive copies of sld-3 are inserted at a MosSCI site and expressed from the mex-5 promoter. n refers to the number of mothers analysed. F. Percentage of progeny from heterozygous +/sld-3(2D) (top) or +/sld-3(2E) (bottom) parents that were either fertile or sterile. These 2D/2E mutations were generated by CRISPR at the endogenous sld-3 locus. For 2D n = 38, for 2E n = 91.