Fig 2: Cell proliferation and DNA replication in the ORC2-/- cell lines.(A) Growth curves of indicated clones of cells over five days, expressed as MTT absorbance relative to the level at day 1. (Mean ± S.D.; n = 4 biological replicates. Cells after passage for three months). (B) FACS profile of propidium-iodide stained cell-cycle asynchronous cells from indicated clones. (C) Cells arrested in double-thymidine block released into nocodazole containing medium and harvested at indicated times after release to measure rate of progression through S phase. AS: asynchronous cells. The red dotted lines indicate cells with G1 and G2 DNA content. (D) Cell-cycle asynchronous cells labeled with BrdU for 30 min. % of BrdU labeled cells evaluated by two color FACS. (Two-sided t-test for two samples, Mean ± S.D.; n = 4 biological replicates). (E) Molecular combing of chromosomal DNA after a pulse of CldU for 30 min chased with a pulse of IdU for 30 min. Top: Schematic shows distances that were measured to estimate fork progression rate and inter-origin distance. Middle: Representative image of the combed DNA stained for CldU (green) and IdU (red) shown below the schematic. Bottom: Box and whiskers plot for fork progression rate and inter-origin distance of indicated clones of cells. (P value < 0.01, two-sided Wilcoxon rank sum test for two samples; N = number of tracks counted. P: Statistical significance of any difference between WT and ORC2-/- cells. (F) Overlap of the BrIP-seq peaks between WT and ORC2-/- cells. (G) Box and whiskers plot for inter-origin distances (measured by BrIP-seq) for each chromosome in WT and ORC2-/- cells. The median inter-origin distance for all chromosomes together indicated at bottom right. (H) Circos plot of Origins mapped by BrIP-seq for chromosome 1. Outer circle: the chromosome with the karytotyping bands. Inner two circles: the locations of BrIPseq peaks in the WT and ORC2-/- cell lines. (I) Distribution of BrIP-seq mapped origins relative to distance from Transcription Start Sites (TSS). In WT and ORC2-/- cells.DOI: http://dx.doi.org/10.7554/eLife.19084.004

Fig 3: In vivo association of ORC2, cdc6 and cdt1 to the chromosomal loci of clones 3, 13 and 32 in normal and transformed cell lines. Histogram plots of the quantification by real-time PCR of immunoprecipitated DNA abundance (ng) at the chromosomal loci of clones 3 (A), 13 (B) and 32 (C) respectively, in two transformed cell lines [HeLa and WI38(SV40)] and two normal cell lines (NSF and WI38). Chromatin IP was performed with antibodies directed against ORC2 (dark gray bars), cdc6 (white bars) and cdt1 (light gray bars); normal rabbit serum (NRS) (black bars) was used as a negative control. The primer sets of clones 3 (A), 13 (B) and 32 (C) are explained in the legend of Figure 3 and Supplementary Table S2. The error bars represent the average of at least two experiments performed in triplicate and 1 SD. (D) Western blot analysis using anti-ORC2, anti-cdc6 and anti-cdt1 antibodies to verify the IP of ORC2, cdc6 and cdt1 proteins in all cell lines used. IP with NRS was used as a negative control.

Fig 4: Verification of the proteins in the complex identified by EMSA and mass spectrometry. Western blot analysis performed on (A) T (electroeluted excised cdc6 supershift bands from transformed NEs) and N (electroeluted excised cdc6 supershift bands from normal NEs) samples as well as (B) T (electroeluted excised cdc6 supershift bands from transformed NEs) and C (electroeluted excised control bands) samples after SDS-PAGE and transfer onto a PVDF membrane to confirm the composition of the transformation-specific complex with the following antibodies: anti-cdc6, anti-EEF1D, anti-Fe65, anti-FKBP-25, anti-ORC1, anti-ORC2, and anti-REF-1. The protein complexes have been analyzed independently a total of 4 times and the experiments were performed from different cell batches giving the same result each time.

Fig 5: Knockout of ORC2 in HCT116 p53-/- cells.(A) Strategy for insertion of a blasticidin gene and poly A site in the fourth exon of ORC2 at aa 40 of ORC2. (B) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC2+/+. ORC2 Knockout clones, B2 and BP8 have an insert on both alleles of ORC2 as indicated by the absence of 0.6 kb PCR product. (C) Verification of antibodies recognizing N-terminal or C-terminal parts of ORC2. Recombinant ORC2 protein halves with Flag epitope tags were expressed and blotted with indicated antibodies. Ponceau S staining of total protein shows equal loading of lanes.* indicates full length endogenous ORC2 protein. Arrow indicates recombinant protein. (D) Quantitative Western blot for ORC2 with an antibody recognizing the N-terminal half of ORC2. Indicated amount of lysate loaded in each lane. (E) Western blot with antibody recognizing C-terminal half of ORC2. * Non specific band (F) Input cell lysate and immunoprecipitates of ORC2 immunoblotted for ORC2. Darker exposure of the top blots is shown in the middle. HSP90 in the cell lysate or the IgG band in the immunoprecipitate serves as loading control. (G) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC2 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. (H) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. Ponceau S staining of histones serves as loading control for chromatin fractions. For each panel, all the lanes are from the same blot and exposure. (I) Comparison of Coomassie Brilliant Blue signal of pure BSA and recombinant purified GST-ORC2 to show that the top-most band in the ORC2 lane is at 170 ng/ 10 µl. (J) Immunoblot with different amounts of cell lysate with the GST-ORC2 to show that 1×10e5 cells give an ORC2 signal equal to 2.54 ng (1.4 fold of 1.67 ng) of GST-ORC2, which corresponds to 153×10e8 molecules of GST-ORC2. (K) Western blot of ORC2 in HBEC and 293T cell lines. Ponceau S staining of total protein or immunoblot of Chk1 show equal loading of the pairs of lanes.DOI: http://dx.doi.org/10.7554/eLife.19084.003