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First published online 7 December 2004
doi: 10.1242/jcs.01599

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Ciz1 promotes mammalian DNA replication

¹ Department of Biology (Area 9), University of York, York, YO10 5YW, UK
² Hutchison/MRC Research Centre, MRC Cancer Cell Unit, Hills Road, Cambridge, CB2 2XZ, UK
³ Heart Research Centre and CRISTAL, Leeds University, Leeds, LS2 9JT, UK

View larger version (43K): [in a new window]   Fig. 1. p100-Ciz1 is co-regulated with cyclin A-cdk2-induced DNA replication. (A) Anti-Cdc6 antibody V detects mouse Cdc6 and a second antigen in western blots of 3T3 whole cell extract, which migrates with an approximate molecular mass of 100 kDa. p100 is present in both the soluble fraction and insoluble nuclear fraction (prepared under in vitro replication conditions). (B) Initiation of DNA synthesis in `replication competent' late G1 phase nuclei by G1 extract supplemented with recombinant cyclin A-cdk2. Control bar shows the proportion of nuclei already in S phase (unshaded), and those that initiated replication in extract from S phase cells (shaded). (C) Late G1 nuclei re-isolated after incubation in parallel reactions with recombinant cyclin A-cdk2 and G1 extract, probed for Mcm2 and Mcm3. (D) p100 antigen is more abundant in nuclei exposed to initiation-inducing concentrations of cyclin A-cdk2, revealed when the same nuclei are probed with antibody V.

View larger version (42K): [in a new window]   Fig. 2. Mouse Ciz1 amino-acid sequence features, exon usage and expression constructs. Antibody V was used to clone the gene for p100 from a mouse embryo expression library (see Materials and Methods), which was identified as Ciz1. (A) Alignment of mouse Ciz1 variants. The predicted full-length Ciz1 amino-acid sequence (Full) is identical to a mouse mammary tumour cDNA clone (BC018483), while embryonic Ciz1 (ECiz1, AJ575057), and a melanoma-derived clone (AK089986) lack two discrete internal sequences. In addition, the first available methionine in ECiz1 (Met84) is in the middle of exon 3, which excludes a polyglutamine rich region from the N terminus. Stars indicate threonine residues changed by site-directed mutagenesis in the constructs shown in D. Amino-acids that correspond to codons targeted by siRNAs are underlined. (B) Mouse Ciz1 is encoded by 17 exons. Coding exons are shown in grey, alternatively spliced regions in mouse ECiz1 are black. (C) Sequence features and putative domains in ECiz1. The C terminal `matrin 3 domain' has homology with the nuclear matrix protein matrin 3 (Nakayasu and Berezney, 1991). The positions of sequences absent from ECiz1 are indicated by triangles. (D) ECiz1 and derived truncations and point mutants used in cell-free DNA replication experiments. Numbers in parentheses relate to amino-acid positions in the full-length form of mouse Ciz1, shown in A. Stars indicate putative phosphorylation sites made unphosphorylatable by site-directed mutagenesis.

View larger version (26K): [in a new window]   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.

View larger version (27K): [in a new window]   Fig. 4. Ciz1 and ECiz1 stimulate S phase entry in wild-type and p21 null cells. (A) ECiz1 and the full-length variant, Ciz1, were tagged with green fluorescent protein to allow identification of expressing cells (green), within transfected populations. Examples of Ciz1-expressing and -non-expressing cells that have or have not engaged in DNA synthesis (and incorporated BrdU into DNA, red) are shown. Nuclei are counterstained with Hoechst 33258 (blue). (B) Expression of ectopic GFP-Ciz1 or ECiz1 in the presence of BrdU increases the number of NIH 3T3 cells that undergo DNA synthesis in rapidly cycling and newly confluent populations, during the17 hour period following transfection. Cells expressing GFP alone are not stimulated to engage in DNA synthesis. (C) Similar results were obtained with p21cip1 null mouse embryo fibroblasts. Histograms show GFP-expressing cells as green bars and non-expressing cells from the same populations as grey bars. Representative results from three independent experiments for each of the conditions are shown. All experiments gave the same effect, but the background fraction of labelled cells in the untransfected population varied according to the density and growth rate of the cells.

View larger version (58K): [in a new window]   Fig. 5. Anti-Ciz1 antibody detects endogenous Ciz1 in sub-nuclear foci that overlap with sites of DNA replication. (A) Coomassie Blue-stained SDS-polyacrylamide gel showing purified recombinant ECiz1 fragment Nterm442, and western blots of recombinant Nterm442 using anti-Cdc6 antibody V, and anti-Ciz1 antibody 1793. (B) Western blot of 3T3 whole cell extract. Of the two bands detected by anti-Ciz1 antibody 1793 one has the same mobility as p100-Ciz1 recognized by antibody V and the other has an apparent molecular mass of 125 kDa. (C) Immunoprecipitation from 3T3 nuclear extract, using antibody V or anti-Ciz1 1793. Both antibodies precipitate p100, which is recognized by the reciprocal antibody in western blots. p125 is precipitated by antibody 1793, and to a lesser extent by antibody V and these are recognized by 1793 in western blots. Mcm3 is shown as a control. (D) Endogenous Ciz1 (red) in 3T3 cells fixed before (untreated) or after (detergent treated) exposure to TritonX100, detected with anti-Ciz1 antibody 1793. Nuclei are counterstained with Hoechst 33258 (blue). (E) Nuclei isolated for replication experiments contain detergent resistant and detergent soluble Ciz1 protein, detected with anti-Ciz1 1793. (F) Detergent-resistant Ciz1 (red) is present in all nuclei in cycling populations, while detergent resistant PCNA (green) persists only in S phase nuclei. (G) High magnification confocal section showing detergent resistant Ciz1 and PCNA foci, and merged image showing co-localising foci (yellow). White arrows indicate some foci common to both antigens. (H) Line plot of red and green fluorescence across the merged image in G, at the position indicated by an arrow. (I) Cross-correlation plot (Rubbi and Milner, 2000; van Steensel et al., 1996) for green foci compared to red over the whole merged image in G, and (inset) for the marked section after thresh-holding fluorescence at the levels shown in H. The red line in the inset shows loss of correlation when the Ciz1 image is rotated 90° with respect to PCNA. Bar is 10 µM.

View larger version (37K): [in a new window]   Fig. 6. Inhibition of new Ciz1 synthesis restrains entry to S phase. (A) Left: siRNAs that target Ciz1 transcripts at two sites (see Fig. 2A) were individually applied to cycling 3T3 cells and cell number was monitored at the indicated times. Right: images of cell populations at 16 and 40 hours after transfection with siRNA 8 (red outline) or mock treated cells (blue outline). (B) The proportion of cells that incorporate BrdU into DNA (green) is significantly decreased in cells treated with Ciz1 siRNAs 4 and 8, compared to GAPDH siRNA, 48 hours after treatment. Histogram shows average results from four independent experiments. (C) The number of nuclei with detergent-resistant Mcm3 and PCNA (green) increases in populations treated with Ciz1 siRNA 4/8, compared with mock-treated controls. All nuclei were counterstained and are shown in pseudocolour (red). (D) Quiescent 3T3 cells were stimulated to re-enter the cell cycle (Coverley et al., 2002) with and without exposure to Ciz1 siRNA 4/8 or GAPDH siRNA, and pulse labelled with BrdU at the indicated times to reveal the proportion of cells in S phase. (E) Quiescent 3T3 cells were stimulated to re-enter the cell cycle and harvested at the G1-S transition (18 hours, black) or after S phase (36 hours in the presence of nocodazole, red) with and without exposure to Ciz1 siRNA 4/8. (F) Detergent-soluble and -insoluble fractions of endogenous Ciz1 protein in approximately 1x105 cells after mock treatment or treatment with Ciz1 siRNAs 4/8, detected with anti-Ciz1 1793. (G) To focus on newly synthesized Ciz1, expression of ectopic GFP-ECiz1 was monitored in whole cell extracts made from approximately 8x103 cells, in the presence and absence of Ciz1 siRNA 4/8 using anti-Ciz1 1793. Expression levels in extracts prepared 22 hours after transfection are shown. For F and G band intensities were quantified (shown in white), normalised against actin levels and expressed as a percentage.

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