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First published online 12 July 2005
doi: 10.1242/jcs.02427

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Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80^+/- cells

Sahar Sibani^1,2, Gerald B. Price^1,* and Maria Zannis-Hadjopoulos^1,2,

¹ McGill Cancer Center, McGill University, Montreal, Quebec H3G 1Y6, Canada
² Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada

View larger version (43K): [in a new window]   Fig. 1. Western blot assay of nuclear extracts (NE) from wild-type (HCT116) and Ku80+/- cells. NE were prepared from HCT116 and Ku80+/- by high-salt extraction of isolated nuclei and were immunoblotted for both subunits of Ku. The expression of p53 and p21 was examined to determine whether they were induced by the reduced Ku80 levels in Ku80+/- cells. Expression of Cdc6 and Cdk2 were also inspected to verify that Ku80 deficiency did not reduce their expression. Nuclear actin was used as a loading control. (A) Increasing amounts (10, 20 and 30 µg) of NE were subjected to electrophoresis and western blotting (as described in Materials and Methods). Immunoblotting with 1/100th dilution of anti-Ku80 (C-20), or 1/1000th dilution of anti-Ku70 (C-19), anti-p53 (FL393), anti-p21 (C19), anti-Cdc6 (D12), anti-Cdk2 (H304), and anti-actin antibody was carried out. (B) Histogram plots of quantifications of the Ku80, Ku70 and actin bands shown in A. Each error bar representing three experiments and one standard deviation (s.d.) is indicated. Signals obtained for HCT116 NE were set at 100%, and those of Ku80+/- were expressed as a percentage of them. An asterisk (*) represents statistically significant differences (P<0.05) in the expression of the indicated protein between HCT116 and Ku80+/- cells.

View larger version (21K): [in a new window]   Fig. 2. Chromatin loading assay of Ku80, Ku70 and MCM7 in HCT116 and Ku80+/- cells. Chromatin-enriched extracts were prepared (as described in Materials and Methods) and subjected to electrophoresis on 5% stacking/8% separating gels and western blotting. The abundance of Ku80 and Ku70 in the chromatin-enriched fractions were assessed. Actin was used a loading control. (A) Immunoblotting with 1/100th dilution of anti-Ku80 (C-20), 1/1000th dilution of anti-Ku70 (C-19) or 1/1000th dilution of anti-actin antibody is shown. (B) Quantification of the Ku80 and Ku70 signals after normalization for actin. Each error bar represents three experiments and 1 SD is indicated. Signals obtained for HCT116 were set at 100% and those of Ku80+/- were expressed as a percentage of them. An asterisk (*) represents statistically significant differences (P<0.05) in the expression of the indicated protein between HCT116 and Ku80+/- cells.

View larger version (33K): [in a new window]   Fig. 3. Flow cytometric analysis of HCT116 and Ku80+/- cells following synchronization to late G1 and release into S-phase. Cells were synchronized to late G1 by thymidine/mimosine double block (late G1), and released into complete medium. They were harvested at 2 (S2), 4 (S4), 6 (S6) and 8 (S8) hours after release, and stained with propidium iodide to monitor their cell cycle distribution. An asynchronous culture of logarithmically growing cells (Log) was used for comparison. (A) Representative data from six experiments are shown. The top panel represents the wild-type (HCT116) cells, and the bottom panel is of the Ku80+/- ones. (B) Quantification of the percentage of cells present in each phase of the cell cycle (G1, S or G2/M) during the synchrony (G1) and release (S2-S8). Averages of six experiments and their standard deviations (in brackets) are shown. Cell cycle phases statistically different (P<0.05) between Ku80+/- and HCT116 cells are shown in bold.

View larger version (37K): [in a new window]   Fig. 4. Abundance of DNA repair proteins Ku80, Ku70 and RPA70 in chromatin-enriched fractions, following exposure of Ku80+/- cells to mimosine or hydroxyurea. Chromatin fractions were prepared from HCT116 and Ku80+/- cells that were either untreated or treated with thymidine/mimosine or thymidine/hydroxyurea double block. (A) Ten and 20 µg were subjected to electrophoresis and transferred onto PVDF membrane. They were then immunoblotted with 1/1000th dilution of anti-Ku80 (H300), 1/1000th dilution of anti-Ku70 (C-19), anti-RPA70, or anti-actin. (B) Quantification of the immunoblots after normalization for actin. Averages of three experiments and 1 s.d. are shown.

View larger version (19K): [in a new window]   Fig. 5. Maps of the lamin B2, ß-globin and c-myc origin loci, and location and characteristics of the LightCycler primers. (A) Maps of the lamin B2 (top left), ß-globin (top right) and c-myc (bottom left) regions. The location and names of the primers is indicated (arrows). For the lamin B2 locus, the exon (black boxes) numbers of the lamin B2 and ppv1 genes are indicated above. For the ß-globin locus, the name of each globin gene (black box) is indicated above. For the c-myc locus, the promoters (arrows) and exons (black boxes) of the c-myc gene are indicated. The distance between the origin-containing and origin-lacking amplicons are indicated in kb (double-headed arrows). (B) The melting curves of the new primers designed for the ß-globin and c-myc origins are shown. Two curves are shown with each primer pair, one containing (+DNA) and one lacking (-DNA) template DNA. None of the primer sets showed any nonspecific products. (C) Standard curves used for the quantification analysis by the LightCycler instrument for each of the four primer sets shown in B. The correlation coefficients (r2) and slopes are indicated. The similarity in the slopes indicates similar primer efficiencies between the different primer pairs.

View larger version (15K): [in a new window]   Fig. 6. Abundance of nascent DNA within the 0.5-1 kb and 1-2 kb DNA fractions at three origins in HCT116. Nascent DNA from an asynchronous culture of HCT116, prepared as described in Materials and Methods, was fractionated on a nondenaturing 1% agarose gel following heat denaturation. Regions containing DNA spanning 0.5-1 kb and 1-2 kb were cut out and purified. Abundance of nascent DNA within those fractions was determined using realtime PCR, at three origins: lamin B2 (A), ß-globin (B) and c-myc (C) origins. As described in Fig. 5, LB2, BG40.9 and Myc11 are the origin-containing regions for these origins, whereas LB2C1, BG72 and Myc1 are distal origin-lacking regions lacking origin activity. The average of three experiments and 1SD is indicated. An asterisk (*) represents a statistically significant difference (P<0.05) between the 0.5-1 kb and 1-2 kb DNA fractions in the abundance of nascent DNA at the specified DNA regions.

View larger version (14K): [in a new window]   Fig. 7. Quantification of nascent strand abundance at three origins in Ku80+/- and wild-type cells. Nascent DNA from the 0.5-1 kb fraction prepared from asynchronous cultures of HCT116 and Ku80+/- cells as in Fig. 5. Origin activity of the lamin B2 (A), ß-globin (B) and c-myc (C) origins were measured by quantification of the nascent strand abundance at each origin. LB2, BG40.9 and Myc11 are located within the origins whereas LB2C1, BG72 and Myc1 are origin-lacking controls, located distant to the origins (see Fig. 4A for maps). Each error bar represents three experiments and 1 SD is indicated. An asterisk (*) represents a statistically significant difference (P<0.05) between HCT116 and Ku80+/- cells in the abundance of nascent DNA at the specified DNA regions.

View larger version (20K): [in a new window]   Fig. 8. Association of Ku70, Ku80 and Orc2 to the lamin B2, ß-globin and c-myc origins in HCT116 cells. Asynchronous cultures of HCT116 cells were treated with 1% formaldehyde and harvested. Chromatin fractions were immunoprecipitated with either anti-Ku80, -Ku70 or -Orc2 antibodies, or normal goat serum (NGS) as control. (A) Western blot analyses of the immunoprecipitates with the immunoblotting antibody indicated on the left and the immunoprecipitating one above the figure. (B) The abundance of DNA for three origins (LB2, BG40.9 and Myc11) along with their origin-lacking controls (LB2C1, BG72 and Myc1) in each of the precipitates was determined using realtime PCR, thus measuring the association of each protein with the examined DNA region. The average of three experiments and 1 SD is shown. An asterisk (*) represents statistically significant differences (P<0.05) in the association of the indicated protein between the origin-containing and origin-lacking regions.

View larger version (21K): [in a new window]   Fig. 9. Chromatin immunoprecipitation (ChIP) assay of HCT116 and Ku80+/- cells at three DNA replication origins and quantification by Real-time PCR. Real-time PCR using the LightCycler instrument was used to quantify the abundance of immunoprecipitated DNA at the origin-containing and origin-lacking regions in the three loci. (A) Quantification of lamin B2 origin-containing (LB2) or -lacking (LB2C1) regions in the three different immunoprecipitates (anti-Ku80, anti-Ku70, and NGS) from 2x107 cells. (B) Quantification of BG40.9 and BG72 DNA regions in Ku80, Ku70 and NGS immunoprecipitates. (C) Abundance of Myc11 and Myc1 DNA in Ku80, Ku70 and NGS immunoprecipitates. For all the bar graphs, the immunoprecipitating antibody is shown on the X-axis, along with the region amplified. Each error bar represents three experiments and 1 SD is indicated. An asterisk (*) represents statistically significant differences (P<0.05) between HCT116 and Ku80+/- cells in the association of the indicated protein to the specified regions.