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Fig. 3. ECiz1 promotes initiation of mammalian DNA replication (A) Recombinant ECiz1 stimulates initiation of DNA replication in `replication competent' late G1 phase nuclei, during incubation in S phase extract. Histogram shows the average proportion of nuclei that initiated DNA replication in vitro (black), in the presence or absence of 1 nM ectopic ECiz1, with standard deviations calculated from four independent experiments. The 17% of nuclei that were already in S phase when the nuclear preparation was made are shown in white. Images show nuclei replicating in vitro, with or without ECiz1. (B-G) The effect of the various recombinant proteins on initiation of DNA replication. (B) The effect of ECiz1 is concentration dependent, with a sharp optimum around 1 nM. (C) Mutation of the predicted cdk phosphorylation site at 191/2 alters the activity profile of ECiz1, so that T(191/2)A mutant remains capable of stimulating initiation even at high concentrations. (D) T(191/2)A (13 nM) functions in the presence of inactive (6 nM) concentrations of ECiz1. (E) Cdk site mutant T(293)A stimulates initiation with a similar profile to ECiz1. (F) Truncated ECiz1 (Nterm 442) lacks C-terminal sequences, but stimulates in vitro initiation to a similar extent as ECiz1. (G) Cterm 274 retains no DNA replication activity in this assay.





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