hi1573 zebrafish embryos show a MAI phenotype. (A) Scheme of checkpoint screen. (B) Whole-mount immunostaining of pH3 showed that IR treatment induces the appropriate block to mitotic entry in 36-hpf wild-type (wt) embryos, but the hi1573 homozygous mutant (mut) clutchmates retained a high mitotic index. (C) Cyclin B1–Cdc2 was immunoprecipitated from 40-hpf nonirradiated (−IR) and irradiated (+IR) pools of wild-type (+/+ and +/−) and mutant (−/−) hi1573 embryos. The total levels of Cdc2 were determined by Western blotting (representative experiment shown). Assessment of cyclin B1–Cdc2 kinase activity (mean ± SD; n = 3 biological replicates) showed that the hi1573 mutants retain high activity after IR treatment. (D) Nonirradiated and irradiated 36-hpf wild-type (+/+ and +/−) and hi1573 mutant (−/−) clutchmates were maintained in the absence or presence of nocodazole for 2 h. The percentage of pH3-positive cells was quantified by FACS (mean ± SD; n = 20,000 cells counted for each of three biological replicates).

Ticrr is required for normal DNA replication and S/M checkpoint function. (A) Cell cycle profile from a representative pool of 40-hpf hi1573 wild-type (+/+ and +/−) and mutant (−/−) zebrafish embryos and quantification of cells with G1, S, or G2/M DNA content (mean ± SD, n = 3 biological replicates) showed an increase in S-phase cells in the ticrr mutants. (B) BrdU and propidium iodide (PI) FACS analysis of cells from pools of BrdU pulse-labeled wild-type and ticrr mutant embryos. Quantification of the BrdU⁺ population (mean ± SD, n = 3 biological replicates) showed a dramatic reduction in replicating DNA in ticrr mutants. (C) Quantification of the proportion of pH3-positive cells in various mitotic phases (mean ± SD; n = 100 cells counted in each of four wild-type and mutant embryos) established a defect in mitotic progression and the presence of abnormal anaphase cells. Representative cells with anaphase bridges are shown. (D) Metaphase spreads of cells from colchicine-treated embryos showed mitotic cells with fragmented chromosomes. A representative wild-type metaphase spread and examples of abnormal metaphase spreads from mutants are shown. (E) FACS measurement of anti-pH3 and PI staining of cells from wild-type and mutant embryos showed a population of mutant pH3-positive cells with less than 4N DNA content (blue) in ticrr mutants, establishing entry into mitosis before completing DNA replication.

Human TICRR is a chromatin-associated protein that interacts with TopBP1. (A) Western blotting of biochemical fractions from HeLa cells showed that the majority of TICRR is present in the chromatin-enriched fraction (chrom) and can be partially released by micrococcal nuclease (Mnase) treatment. Orc2 and α-tubulin are chromatin and cytoplasmic markers. (WCE) Whole-cell extract; (S1) soluble cytoplasmic fraction; (S2) soluble nuclear fraction. (B) Immunoblotting showed TopBP1 coimmunoprecipitates with TICRR but not preimmune (PI) antibodies. The association was not affected by treatment with ethidium bromide (EtBr) or DNase I, indicating its independence from chromatin binding. (C) Reciprocal immunoprecipitation-Western blotting confirmed that TICRR is present in TopBP1, but not IgG, immunoprecipitates. (D) Immunoprecipitation-Western blotting showed that GFP-tagged zebrafish Ticrr associated with human TopBP1 when coexpressed in human cells. (E) Anti-GFP immunoprecipitates and whole-cell lysates (input) from cells transfected with GFP-tagged TopBP1 deletion mutants were screened for TICRR and TopBP1 proteins by immunoblotting. (F) HeLa cells were synchronized in mitosis and then allowed to re-enter the cell cycle in the absence (−Rosc) or presence (+Rosc) of roscovitine as depicted. FACS analysis showed that the −Rosc cells were entering S phase, while the +Rosc cells remained in G1. Immunoprecipitation-Western analysis shows that the TICRR–TopBP1 complex was recovered at similar levels in the −Rosc and +Rosc samples. (AS) Asynchronous. (G) Asynchronous HeLaS3 cell extracts were immunoprecipitated with anti-TICRR antibodies and then incubated with either λ phosphatase, phosphatase inhibitors, or both. The mobility of the TICRR protein was significantly increased in the phosphatase-treated sample versus the controls, but showed no difference in the levels of associated TopBP1.

The checkpoint defect results from disruption of a novel gene, ticrr. (A) Schematic of the zebrafish ticrr gene denoting the position of viral insertions in the hi1573 and hi3202A mutant lines. (B) Analysis of pH3 showed a low mitotic index in wild-type embryos but not hi3202A mutant clutchmates 1 h after IR exposure, showing that hi3202A also has a checkpoint defect. (C,D) Quantification of total mRNA by RT–PCR showed that the levels of ticrr mRNA were greatly reduced in both the hi1573 (C) and hi3202A (D) homozygous mutants at 36 hpf.

A DNA replication defect but not premature mitotic entry occurs in 24-hpf ticrr mutants. (A) Cell cycle profiles from 24-hpf hi1573 wild-type (+/+) and mutant (−/−) zebrafish embryos and quantitation of cells with G1, S, or G2/M DNA content (mean ± SD, n = 3 biological replicates), showed an increase in S-phase cells in mutants. (B) BrdU and PI FACS analysis of cells from pools of BrdU pulse-labeled, 24-hpf wild-type and ticrr mutant embryos. Quantification of the BrdU⁺ population showed no change in the percentage of cells replicating DNA (mean ± SD, n = 20,000 cells in each of three biological replicates), but the level of BrdU incorporation per cell is significantly decreased in the mutants (mean BrdU signal = Mean[BrdU⁺ signal]/Mean[BrdU− signal]; Student's t-test P < 0.05). (C) FACS measurement of anti-pH3 and PI staining of cells from 24-hpf wild-type and mutant embryos showed no evidence of an S/M defect in the ticrr mutants at this time point, as judged by the absence of pH3-positive cells with less than 4N DNA content.

Ticrr is essential for the chromatin association of the pre-IC component Psf1. Total cell lysates and chromatin-enriched fractions were prepared from 40-hpf wild-type (+/+ and +/−) and ticrr mutant (−/−) zebrafish embryos. Immunoblotting for core pre-RC and pre-IC components, the MCMs and Psf1, respectively, showed that these are expressed at normal levels, but only the MCMs are chromatin-loaded, in the ticrr mutants.