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Two subunits of human ORC are dispensable for DNA replication and proliferation

  1. Etsuko Shibata
  2. Manjari Kiran
  3. Yoshiyuki Shibata
  4. Samarendra Singh
  5. Shashi Kiran
  6. Anindya Dutta  Is a corresponding author
  1. University of Virginia School of Medicine, United States
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Cite this article as: eLife 2016;5:e19084 doi: 10.7554/eLife.19084

Abstract

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1. The ORC1 or ORC2-depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication

https://doi.org/10.7554/eLife.19084.001

eLife digest

Most of the DNA in human cells is packaged into structures called chromosomes. Before a cell divides, the DNA in each chromosome is carefully copied. This process begins at multiple sites (known as origins) on each chromosome. A group of six proteins collectively known as the Origin Recognition Complex (or ORC for short) binds to an origin and then recruits several additional proteins. When the cell is ready, the assembled proteins are activated and DNA copying begins. It is thought that all of the ORC proteins are essential for cells to survive and copy their DNA.

Here, Shibata et al. reveal that human cells can survive without ORC1 or ORC2, two of the six proteins in the ORC complex. Disrupting the genes that encode the ORC1 and ORC2 proteins in human cancer cell lines had little effect on the ability of the cells to copy their DNA and survive. Furthermore, these cells spend the same amount of time copying their DNA and use a similar set of origins as normal cells.

However, the experiments also reveal that cells without ORC1 or ORC2 are more dependent on the presence of one particular protein recruited to the origin after the ORC assembles. Reducing the availability of this protein, CDC6, decreased the ability of these cells to survive and divide. Future efforts will aim to identify the mechanism by which cells bring together the proteins required to copy DNA in the absence of a complete ORC.

https://doi.org/10.7554/eLife.19084.002

Introduction

The discovery of the six-subunit ORC (Bell and Stillman, 1992) identified the long sought initiator protein that binds to replicator sequences to initiate DNA replication in eukaryotes. ORC is an essential six-subunit, ring-shaped ATPase complex that recruits and co-operates with the CDC6 protein to promote the loading of CDT1 and then the MCM2-7 subunits of the replicative helicase during the ‘licensing’ of origins of replication (Bleichert et al., 2015; Yeeles et al., 2015; Bell and Stillman, 1992 Blow and Tada, 2000; Masai et al., 2010). Of the six subunits of human ORC, ORC2-5 form a tight core complex (Dhar et al., 2001). ORC1 is the only subunit responsible for the ATPase activity of ORC (Chesnokov et al., 2001; Giordano-Coltart et al., 2005; Klemm et al., 1997) All six ORC subunit genes are essential for the viability of S. cerevisiae and S. pombe (Bell et al., 1993; Foss et al., 1993; Herskowitz, 1993; Loo et al., 1995; Micklem et al., 1993). It is therefore expected that eukaryotic cells will not be viable, and will not replicate, if any of the ORC subunit genes are deleted. However, we have now deleted both alleles of human ORC2 or of ORC1, to discover that cells can still survive and replicate in the complete absence of either of these two critical subunits of Origin Recognition Complex.

Results

Biallelic disruption of the ORC2 gene

CRISPR/Cas9 was used to insert a ~600 bp blasticidin gene and poly A site into exon 4 (amino acid 40) of ORC2 in HCT116 p53-/- (Bunz et al., 1998) colon cancer cells (WT: HCT116 p53-/-, ORC2+/+) (Figure 1A and B). All clones were viable and proliferated for months despite the disruption of the ORC2 gene. Immunoblotting and immunoprecipitation-immunoblotting of cell extracts showed that the ORC2-/- B2 and BP8 clones had no detectable ORC2 protein (Figure 1D–H). An anti-ORC2C antibody recognizing the C terminal half of ORC2 (Figure 1C) ensured that a C-terminal fragment of ORC2 was not being expressed from an alternatively spliced transcript using an internal methionine (Figure 1E). Quantitative immunoblotting showed that if any ORC2 was expressed, it must be at a level <1% of wild type levels (Figure 1D). Quantitative immunoblotting of cell lysates and carefully measured amounts of recombinant bacterially produced ORC2 fragment showed that wild type cells express ~153,000 ORC2 molecules per cell (Figure 1I,J). Thus, if the B2 or BP8 clones contain any ORC2 molecules below the level of detection, they can have no more than 1530 molecules/cell. ORC2 was also deleted in 293T human embryonic kidney cells or HBEC human bronchial epithelial cells immortalized with CDK4 and hTERT, and these cells too continued proliferating in the absence of ORC2 protein (Figure 1K).

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.

https://doi.org/10.7554/eLife.19084.003

CDC6, CDT1 and MCM2-7 loading on chromatin in the absence of ORC2

The ORC2-/- cells suffer a decrease of ORC3, ORC4 and ORC5, which are destabilized when not complexed with ORC2 (Figure 1G). ORC1 was also decreased, but ORC6, CDC6 and CDT1 were unchanged. There was no activation of the DNA damage checkpoint, measured by the phosphorylation of Chk1 or H2AX, as would have occurred with impaired DNA replication. Chromatin association of ORC2-5 and of ORC1 was decreased in ORC2-/- cells (as expected from the decrease of these proteins in cell lysates) (Figure 1H). The chromatin association of MCM3, 5 and 7 of the MCM2-7 was reduced, but not completely eliminated. Surprisingly, chromatin association of ORC6 or CDT1 was relatively unchanged, while CDC6 association was slightly increased.

Initiation of DNA replication in ORC2-/- cells

The proliferation rate of the ORC2-/- cells was >50% that of WT cells (Figure 2A) and ORC2 did not re-appear even after passage of the cells for over a year. The ORC2-/- cells did not accumulate in S phase (Figure 2B) and completed DNA replication after release from an early S-phase block in the same time as WT cells (Figure 2C). The percentage of cells synthesizing DNA during a 30 min pulse was not significantly decreased (Figure 2D). By molecular combing the median distance between bi-directional origins of replication in ORC2-/- cells was marginally increased to 113–118 kb from 96 kb and the fork progression rate was slightly increased to 1.3–1.5 kb/min from 1.2 kb/min (Figure 2E). Given the total DNA content of six billion bp in these cells, this measurement suggests that the ORC2-/- cells fire about 52,000 origins of replication.

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.

https://doi.org/10.7554/eLife.19084.004

We mapped replication initiation sites by a second method: enriching for BrdU-labeled, origin-centered nascent strands by immunoprecipitation and sequencing (BrIPseq) (Karnani et al., 2010). ~13,000 BrIPseq origins were mapped to unique DNA sequence in the ORC2-/- cells compared to ~20,000 in WT cells (Figure 2F). Taking into consideration repetitive DNA and the diploid nature of the human genome, this method also suggests that about 52,000 origins are fired in the ORC2-/- cells. 40% of the BrIP-seq peaks in ORC2-/- cells overlapped with that in WT cells, comparable to the overlap reported among origins mapped by different groups (Karnani et al., 2010). The <100% of overlap is explained by plasticity of origin usage (Cadoret et al., 2008). The inter-origin distances for the BrIPseq origins in ORC2-/- cells (26 kb) were slightly longer than in the WT (25 kb) cells (P value ~4.3e-07, Wilcoxon rank sum test) and the chromosome-by-chromosome distribution of inter-origin distances was similar to that in WT cells (Figure 2G). The 4-fold shorter inter-origin distance in a population-based approach of origin mapping (BrIPseq) compared to molecular combing is also due to the plasticity of origin usage: only one out of four possible origins in a single DNA segment fire in one S phase, while all four origins are used in the entire population of cells (see discussion in Karnani et al., 2010). Origins are enriched in gene-rich domains and near transcription start sites (Figure 2H and I) as reported in previous studies (Karnani et al., 2010; Cadoret et al., 2008; Danis et al., 2004; MacAlpine et al., 2004; Mesner et al., 2011; Sequeira-Mendes et al., 2009).

Survival of ORC1-/- cells

We next disrupted the only subunit of ORC demonstrated to have ATPase activity, ORC1 (Chesnokov et al., 2001; Giordano-Coltart et al., 2005; Klemm et al., 1997). A blasticidin or a plasmid-derived DNA fragment was inserted in exon 1 of ORC1 at the initiator methionine (Figure 3A,B). Western blotting and immunoprecipitation-western blotting of ORC1 protein showed no ORC1 protein in the ORC1-/- clones (Figure 3C–E). ORC1 is loosely associated with the other subunits of ORC, and unlike ORC2, ORC1 depletion did not decrease ORC2, ORC3, ORC4, ORC5, ORC6, CDT1, and CDC6 (Figure 3C). Although ORC1 was absent, the remaining five subunits of ORC along with CDC6 and CDT1 (at least in one clone) could associate with chromatin. However, the chromatin loading of MCM3, 5, and 7 subunits of MCM2-7 is significantly decreased (Figure 3D).

Knockout of ORC1 in HCT116 p53-/- cells.

(A) Strategy for insertion of a blasticidin gene and poly A site after first methionine of ORC1 in the second exon. (B) PCR on genomic DNA of indicated clones. WT: HCT116 p53-/- and ORC1+/+. ORC1 Knockout clones, B14, B48 and BP32 have an insert on both alleles of ORC1 as indicated by the absence of 0.6 kb PCR product. (C) Western blot for indicated proteins in clones indicated on the top. Darker exposure of the ORC1 blots is shown at the bottom. Ponceau S stains all proteins on the blot and also indicates equal loading of lanes. (D) Immunoblot of soluble and chromatin-associated proteins in the clones indicated at the top. For each panel, all the lanes are from the same blot and exposure. (E) Input cell lysate and immunoprecipitates of ORC1 immunoblotted for ORC1. Darker exposure of the top blots is shown in the middle. Tubulin in the cell lysate or the IgG band in the immunoprecipitate serves as loading control.

https://doi.org/10.7554/eLife.19084.005

DNA replication in ORC1-/- clones

At early passage (at one month), the ORC1-/- clones proliferated at 10–20% of the rate of WT cells but by 6 months of passage, their proliferation rates at about 50% of WT cells without reappearance of ORC1 protein (Figures 4A and 3E). The cells did not accumulate in S phase (Figure 4B) and passage through S phase was not slowed (Figure 4C). By molecular combing, inter origin distance in ORC1-/- cells was unchanged or slightly decreased (Figure 4D). Fork progression was decreased to 0.8–1.0 kb/min from 1.2 kb /min. BrIPseq analysis showed that the ORC1-/- cells fire ~13,000 unique origins, with 43% of them overlapping with origins in WT cells (Figure 4E). The distribution of inter-origin distances between chromosomes, enrichment of origins in gene-rich segments and near transcription start sites was similar to that of WT cells or ORC2-/- cells (Figure 4F–H).

Cell proliferation and DNA replication changes in the ORC1-/- cell lines.

(A) Growth curves of indicated clones of cells over four days, expressed as MTT absorbance relative to the level at day 1. (Mean ± S.D.; n = 4 biological replicates) Cells after passage for 1 month or six 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) Molecular combing of chromosomal DNA after a pulse of CldU for 30 min chased with a pulse of IdU for 30 min. Box and whiskers plot for fork progression rate and inter-origin distance of indicated clones of cells. (P value < 4.6e-06, two-sided Wilcoxon rank sum test for two samples N = number of tracks counted) (Inter origin disntance N = 91(WT), 131(ORC1B14), 174(ORC1BP32) p: Statistical significance of any difference between WT and ORC1-/- cells. (E) Overlap of the BrIP-seq peaks between WT and ORC1-/- cells. (F) Box and whiskers plot for inter-origin distances (measured by BrIP-seq) for each chromosome in WT and ORC1-/- cells. The median inter-origin distance for all chromosomes together indicated at bottom right. (G) 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 ORC1-/- cell lines. (H) Distribution of BrIP-seq mapped origins in ORC1-/- cells relative to distance from Transcription Start Sites (TSS).

https://doi.org/10.7554/eLife.19084.006

CDC6 becomes more important for replication in ORC1-/- or ORC2-/- cells

The increase in chromatin association of CDC6 in the ORC2-/- cells (Figure 1H) led us to test whether CDC6 acts inefficiently to load enough MCM2-7 in the absence of the six-subunit ORC and the ATPase subunit of ORC (in the ORC1-/- cells). Knocking down CDC6 in WT cells (Figure 5A) did not decrease either the % of cells in active S phase (Figure 5B) or colony formation (Figure 5C). In contrast, knockdown of CDC6 in the ORC1-/- or ORC2-/- cells increased phosphoChk2, suggesting the activation of DNA damage checkpoints indicating problems in S phase (Figure 5A), decreased actively replicating cells, and decreased colony formation (Figure 5B–C), suggesting that CDC6 becomes more important for DNA replication and cell proliferation in the absence of ORC2 or ORC1.

CDC6 is more essential for replication and colony formation in the ORC mutant cells.

(A) Immunoblots of extracts from indicated cell lines following transfection of siGL2 (negative control siRNA against luciferase) or siCDC6. (B) % of BrdU+ cells after transfection of indicated siRNAs. Data from two color FACS. (P value < 0.01, two-sided t-test for two samples, Mean ± S.D. n = 4 or 3 biological replicates) (C) Top: 72 hr after transfection of indicated siRNAs, 2000 cells were plated per plate for colony formation detected by Crystal violet staining after seven days. Bottom: Crystal violet stained colony density were measured. Data presented for each cell line normalized to the density of the siGL2 transfected cells. (P value < 0.001, two-sided t-test for two samples, Mean ± S.D. n = 3 biological replicates). (D) Immuno blots of ORC5 (E) FACS profile of propidium-iodide stained cells for cell-cycle determination at three days after transfection of indicated siRNA. (F) % of BrdU+ cells after transfection of indicated siRNAs. Data from two color FACS. (Two-sided t-test for two samples, Mean ± S.D. n = 3 biological replicates).

https://doi.org/10.7554/eLife.19084.007

ORC5 protein remains important for DNA replication in ORC1-/- or ORC2-/- cells

To test whether any of the other subunits of the six-subunit ORC remain important for replication in the ORC1-/- or ORC2-/- cells, we depleted ORC5 by siRNA (Figure 5D). Knockdown of ORC5 did not change the cell cycle profile in ORC-/- mutant cells (Figure 5E), and in fact more severely repressed the % of actively replicating cells in WT cells than in the ORC1-/- or ORC2-/- cells (Figure 5F). However knockdown of ORC5 still inhibited BrdU incorporation in the ORC1-/- and ORC2-/- cells. We have not yet succeeded in knocking out the ORC4 and ORC5 genes, though of course such clones may emerge in future attempts. At the present state, our results suggest that the other subunits of ORC may support DNA replication independent of ORC1 or ORC2.

Discussion

The surprising results from this genetic investigation of ORC in human cell lines suggest that two subunits of ORC are dispensable for DNA replication. Although we believe that no ORC2 protein is synthesized in the ORC2-/- cells, we have to entertain the caveat that up to 1530 ORC2 molecules can escape the limits of detection with our current antibodies. This number is still too few to license the 52,000 origins of replication mapped by two independent methods. It is unlikely that a single ORC complex can catalytically load thirty-five MCM2-7 hexamers distributed 100 kb apart over 3.5 Mb of chromosomal DNA.

Since knockdown of ORC5 still affects DNA synthesis in the ORC1-/- or ORC2-/- cells, we cannot yet conclude that replication initiation can occur in the absence of all subunits of ORC. To reach that conclusion we have to successfully delete both alleles of the other four subunits, ORC3-6. However, even if the remaining ORC subunits are functional for loading MCM2-7 our result requires reconsideration of existing models of ORC function in replication initiation. (1) The crystal structure suggests that ORC2-3-5-4-1 are arranged in a gapped ring (with a central channel that is wide enough to surround a DNA double-helix), and that later in licensing, CDC6 slips into the gap between ORC2 and ORC1 to close the gap. The ORC-CDC6 ring is proposed to interact with the MCM2-7 ring end-on-end during the loading of MCM2-7 (Bleichert et al., 2015). Loss of one subunit in the five-membered ORC ring makes it difficult for the remaining subunits to form a ring large enough (i) to surround a DNA double-helix in the same manner as wild type ORC or (ii) to interact with the MCM2-7 ring end-on-end. (2) Human ORC1 and ORC4 are the only subunits that have intact Walker A and B motifs. Multiple groups have shown that the ATPase activity of ORC (in S. cerevisiae, in D. melanogaster and in H. sapiens) depends exclusively on the Walker A and B motifs of the ORC1 subunit, and that this ATP binding and hydrolysis activity is essential for ORC function (Chesnokov et al., 2001; Giordano-Coltart et al., 2005; Klemm et al., 1997). Even if an altered or partial ORC is initiating replication, we have to conclude that any ATPase activity necessary can be provided by ORC4 or CDC6.

There is a report arguing that ORC1 is not essential for endoreplication in Drosophila, because ORC1-/- larvae still allowed endoreplication in salivary gland cells, with only a twofold reduction of ploidy (Park and Asano, 2008). However the paper did not show the sensitivity of the western-blot to measure the level of residual ORC, so that it is theoretically possible that there was enough residual maternal ORC in the endoreplicating cells.

The classic model of replication initiation where ORC first associates with the DNA, helps load CDC6, which then helps load CDT1 and MCM2-7 may still be important for efficient MCM2-7 loading. The surprise is that inefficient MCM2-7 loading, perhaps with the help of CDC6, is sufficient for replication initiation and cell survival in the absence of six-subunit ORC. The other surprise is that the six-subunit ORC does not appear to associate with chromatin as a holocomplex. Clearly ORC6 associates with chromatin normally despite a decrease in ORC1 (in the ORC2-/- or ORC1-/- cells) or ORC2-5 loading (in the ORC2-/- cells). Similarly ORC2-5 loading is independent of ORC1 loading (in the ORC1-/- cells).

The origin plasticity of eukaryotes is attributed to the loading of excess subunits of MCM2-7 on chromatin. It is thus also surprising that the plasticity persists despite a significant reduction in the association of MCM2-7 on chromatin. In summary, we suggest that the absolute requirement for six-subunit ORC for licensing bi-directional origins of replication can be bypassed in some cell lines.

Materials and methods

Cell culture and transfection

HCT116 p53−/− cells (Bunz et al., 1998) (RRID:CVCL_S744, a generous gift from Fred Bunz, Johns Hopkins) were maintained in McCoy's 5A-modified medium supplemented with 10% fetal bovine serum. HBEC3 (RRID:CVCL_X491)-p53KD-K-RasV12 cells (a generous gift from, David R. Jones, Memorial Sloan-Kettering Cancer Center) were originally engineered to express sh-p53 RNA and K-Rasv12 protein by John D. Minna’s group in the University of Texas Southwestern Medical Center (Sato et al., 2006) and were maintained in Keratinocyte-SFM. HBEC3-p53KD-K-RasV12 cells we used also express shGL2 and GFP (Hall et al., 2014). Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) was used to transfect plasmids and RNAiMAX (Thermo Fisher Scientific) was used to transfect siRNAs according to the manufacturer’s protocol. CDC6 siRNA (GAUCGACUUAAUCAGGUAU), ORC5 siRNA (CCCUGGUUGGCCAUGACGA) was synthesized by Thermo Fisher Scientific, Waltham, MA. HEK293T cells (RRID:CVCL_0063) were from ATCC (CRL-3216). No mycoplasma contamination was found. 293T cells and HCT116 p53-/- cells were authenticated by STR profiling.

Plasmids

gRNA was cloned into pCR-Blunt II-TOPO vector backbone (Addgene 41820, Cambridge, MA) using PCR and In-Fusion cloning (Clontech, Mountain View, CA). gRNA target sequence was as follows. GAAGGAGCGAGCGCAGCTTTTGG. Human codon optimized Cas9 nuclease (hCas9) expression vector was obtained from Addgene (41815).

Construction of vectors for homologous recombination

Blasticidin or Hygromycin resistance genes terminated by a polyA sequence and flanked by two homology arms (0.9 kb–1.6 kb in length) were cloned into pDONR 221 (Thermo Fisher Scientific) using PCR and In-Fusion cloning (Clontech).

DNA combing assay

Cells were pulse labeled with 100 µM CldU for 30 min, following by 250 µM IdU for 30 min before embedding into agarose plug. DNA was combed on silanized coverslips (Genomic Vision, Bagneux, France), dehydrated at 65°C for 4 hr and denatured in 0.5 M NaOH and NaCl for 8 min. CldU or IdU were immune-detected with either anti-BrdU antibody that recognizes CldU (MA 182088, Thermo Fisher Scientific, Waltham, MA, RRID:AB_927214) or anti-BrdU antibody that recognizes IdU (347580, BD Biosciences, Franklin Lakes, NJ, RRID:AB_400326). Image acquisition was performed with Zeiss AxioObserver Z1, 63 X objective. DNA lengths were measured using Image J software.

BrdU incorporation

BrdU incorporation was conducted as previously described (Machida et al., 2005) with the following modifications. Cells were labeled with 10 μM BrdU for 30 min and fixed in 70% Ethanol. DNA was denatured in 2 M hydrochloric acid and stained with FITC-conjugated BrdU antibody (556028, BD Biosciences, RRID:AB_396304) and propidium iodide (Sigma-Aldrich, St.Louis) according to the manufacturer's instruction.

Clonogenic assay

To determine the effects of CDC6 knock down, cells were transfected with siRNA twice. 48 hr after the first siRNA transfection, 2000 cells were plated in six well plates. Colonies were fixed, stained with crystal violet, and counted 1 week later. All experiments were conducted in triplicate.

Proliferation assay

1000 cells were plated in 96 well plates. The absorbance of cells were measured every 24 hr using CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, WI) according to the manufacturer's instructions. All experiments were conducted in triplicate and absorbance relative to that on day one was expressed.

Immunoprecipitation, western blot, antibodies, and recombinant protein

Cells were lysed in lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5 % NP-40, 1 mM DTT, 20 mM NaF, and protease inhibitor cocktail. Lysate was cleared by centrifugation and incubated with ORC1(Machida et al., 2005) or ORC2 (Dhar et al., 2001) antibody for 4 hr. Immunoprecipitate was collected on Dynabeads Protein G (Thermo Fisher Scientific) and eluted with 2 x SDS sample buffer. Antibodies used were as follows. ORC2 (Dhar et al., 2001) (Figures 1F, G, H,and and K), ORC2N (Figures 1C, D,and and J) (sc-32734, Santa Cruz Biotechnology, Dallas, TX, RRID:AB_2157726), ORC2C (Figure 1C and E) (sc-13238, Santa Cruz Biotechnology, RRID:AB_2157715), MCM3 (sc-9850, Santa Cruz Biotechnology, RRID:AB_2142269), HSP90 (sc-13119, Santa Cruz Biotechnology, RRID:AB_675659), Cdt1 (Senga et al., 2006), ORC3, ORC4, ORC5, and ORC6 (Machida et al., 2005), ORC1 (4731, Cell Signaling Technology, Danvers, MA, RRID:AB_2157583), CDC6 (3387, Cell Signaling Technology, RRID:AB_2078525), p-Chk1 (2341, Cell Signaling Technology, RRID:AB_330023), p-Chk2 (2661, Cell Signaling Technology, RRID:AB_331479), Chk2 (3440, Cell Signaling Technology, RRID:AB_2229490), p-H2AX (2577, Cell Signaling Technology, RRID:AB_2118011), and H2AX (2595, Cell Signaling Technology, RRID:AB_10694556), MCM5 (A300-195A, Bethyl Laboratories, Inc Montgomery, TX, RRID:AB_185552), MCM7 (A300-128A, Bethyl Laboratories, Inc., RRID:AB_2142821), FLAG (F1804, Sigma, RRID:AB_262044), and Chk1 (NB100-464, Novus Biologicals, LLC, Littleton, CO, RRID:AB_10002158). GST tagged ORC2 recombinant protein was purchased (H00004999-P01, Abnova, Taipei City, Taiwan)

Br-IP Seq

The cells were labelled with 100 µM BrdU (Sigma) in exponential phase of their growth (50–60% of confluency) for 1 hr. The cells were lysed and genomic DNA was isolated, denatured and nascent strands were separated on neutral sucrose gradient. The fragments of 0.5 to 3.0 kb were selected. After dialysis against TE, the DNA was sheared, denatured and immune precipitated with anti-BrdU antibody (B8434, Sigma, RRID:AB_476811). The single stranded BrdU immunoprecipitate (10 ng) was then used to prepare next generation sequencing libraries using Takara Chip-Seq kit. The library from the control genomic DNA was prepared the same way as for the BrdU sample but the only difference was that sucrose gradient centrifugation and size selection was not done for the genomic control. Also the BrdU incorporation was for a longer time (36 hr compared to 1 hr for Br-IP sample). Single-end 75 bp reads were obtained for wildtype (WT) and ORC2 or ORC1 knockout cells. BrdU incorporated genomic strands was also sequenced and used as control (CNTL). Perl script was written and used to trim T’s present at the 3’ end of reads. The trimmed reads were aligned to hg38 using Bowtie2 with the default parameters. Alignment with Bowtie2 resulted in 12597288 (81%) and 10204177 (77%) and 13171609 (93%) mapped reads in WT, KO and CNTL respectively. To define peaks, the genome was divided into 1 kb windows. Any 1 kb window in WT or KO cells with two fold more reads than CNTL were considered to calculate mean and SD. 1 kb windows with reads ≥ mean + 3 SD number of reads were defined as peaks in WT or KO cells. Dataset for transcription start sites (TSSs) was downloaded from the UCSC genome browser. Circos v0.67 (Krzywinski et al., 2009) was used to construct circular genome visualizations. Peaks coordinates of WT and KO chromosome one was parsed to create files appropriately formatted for input to Circos.

Chromatin fractionation

Chromatin fractionation was performed as previously described (Méndez and Stillman, 2000). Cells were resuspended in buffer A (10 mM HEPES [pH7.9], 10% glycerol, 1 mM DTT, protease inhibitor cocktail [Thermo Fisher]). After adding 0.1% Triton X-100, cells were incubated for 5 min on ice and centrifuge at 1300 g, 4°C. Supernatants were further clarified by centrifugation at 20000 g, 4°C (S2). Pellets (Nuclei) were washed in buffer A and lysed in buffer B (3 mM EDTA, 0.2 mM, EGTA, 1 mM DTT, protease inhibitor cockatil). After incubation for 30 min on ice, lysate was centrifuged at 1700 g, 4°C. Pellets (chromatin) were washed in buffer B and lysed in 2 x SDS sample buffer and sonicated (P3).

Statistical analysis

We have used the pwr package in R for choosing sample size for all the experiments. Owing to the nature of the performed experiments, no randomization and no blinding were used. All test statistics were calculated with R (http://www.r-project.org/). t-test and Wilcoxon–rank sum test was performed to test the difference in mean for the normal and skewed data respectively.

Data deposition

Source data including BrIP seq data were deposited in Dryad (Shibata et al., 2016).

References

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Decision letter

  1. Kevin Struhl
    Reviewing Editor; Harvard Medical School, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "ORC is dispensable for DNA replication initiation, but essential for repression of Rb- and polycomb-regulated genes" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

All reviewers find the work potentially important, but to validate that ORC is not essential for DNA replication additional work is required as stated below. Additionally, the experiments suggesting that the ORC complex is important for expression of Rb- and Polycomb target genes is based on single experiments. We suggest that you expand on these experiments or resubmit a shorter version focusing exclusively on the DNA replication part.

Essential revisions:

We all agree that the statement based on the deletion of ORC1 and ORC2, while suggestive of the conclusion, does not provide a definitive answer for ORC-independent DNA replication. We would like to see a deletion or knock down of ORC5.

We look forward receiving a modified version of the studies.

Reviewer #1:

In this manuscript, the authors aim to address the roles of ORC2 and ORC1 proteins in replication by knocking out these proteins in HCT116 and other cell lines. They observe no drastic effects on DNA replication, cell cycle progression, origin number and distribution. They also find that CDC6 plays an important role in this context and that a large number of PRC2 and Rb -dependent genes are deregulated.

Major comment:

Although these findings are important, they do not offer much novel insights. The authors have previously shown a number of the current findings in a hypomorphic ORC2 cell line (JBC (10), 6253-6260). Also, that ORC has roles in gene silencing also has been shown (Genes & Dev (9), 911-924 / Cell (91), 311-323). Additional data that provide some novel mechanistic insight into why ORC is dispensable for replication or what kind of role it plays in transcriptional regulation of PRC2 and Rb regulated genes are necessary to justify publication of this manuscript in eLife.

Reviewer #2:

The manuscript by Dutta and colleagues describes the analysis of deletions of ORC2 or ORC1, key subunits of the origin-recognition complex (ORC), in human cells. ORC is an essential replication protein in simple eukaryotes where origins of replication are defined by specific sequences. In contrast, higher eukaryotes lack a consensus sequence that defines origins and the molecular determinants of the sites of replication initiation in these organisms remain murky. The authors make the intriguing finding that deletions of either the ORC1 or the ORC2 gene are not lethal in human tissue culture cells. They go on to show that loading of the essential Mcm2-7 helicase occurs at reduced levels in these strains but residual loading is observed. Because the chromatin association of Cdc6 and Cdt1, two other helicase-loading proteins is unaffected, the authors suggest that continued association of these proteins mediates the residual Mcm2-7 loading. Intriguingly, depletion of Cdc6 reduced but did not eliminate replication in these cells (perhaps due to incomplete depletion).

The authors do a nice job of showing that deletions of ORC1 and ORC2 are viable and maintain relatively normal patterns of replication. The authors have also done well to show that their deletions are complete and there is not large amounts the proteins retained in the targeted cells. What is less clear is the method by which the residual Mcm2-7 is being loaded. Clearly Cdc6 is still playing a role but whether the remaining ORC subunits are capable of assisting with this is not clear. It would significantly strengthen the paper to show that siRNA of other ORC subunits does not have the same effect as there is either substantial (ORC1 deletion) or residual (ORC2 deletion) binding of other ORC subunits in each case. Overall, this part of the paper convincingly shows that there are other pathways than the canonical ORC-dependent ones to initiate replication.

The authors also observe that the loss of ORC causes changes in gene expression patterns that are similar to E2F1 over-expression strains, Rb knockdown strains and PRC2 subunit deletion strains. While these findings indicate that deletion of ORC subunits leads to changes in the patterns of gene expression, whether these changes are direct or indirect is unclear. The authors suggest that ORC is "critical for the regulation of these genes," however, they do not discuss the possibility that the changes in cell cycle progression as a possible reason for these changes. It would improve the paper to have a more complete discussion of the various reasons that these genes could be altered.

One other point that merits additional discussion is the selection that is going on as they grow up cells after deletion of ORC1 or ORC2. The authors point out that over time these cells are dividing faster which strongly suggests that there are additional genetic changes that alow improved cell growth. This means that the cells being analyzed are not simply ORC1-/- cells or ORC2-/- cells. It is important that the authors acknowledge this caveat and discuss what types of changes could be responsible for the improved growth.

Overall, the authors provide data that strongly suggests that there are non-canonical methods to initiate replication in the absence of ORC1 or ORC2. This is an important and surprising insight into the mechanism of initiation in mammalian cells and is consistent with previous data from this lab and in Drosophila that there could be ORC-independent mechanisms for replication initiation. It remains to be demonstrated whether the residual DNA replication observed is independent of the other ORC subunits (that is truly ORC- independent) or not (this would require double mutants in ORC – it would be a very nice addition if the authors showed that depletion/elimination of a second ORC subunit in the ORC1-/- or ORC2-/- cells did or did not further reduce replication). The gene expression data is relatively underdeveloped and the strong statements made by the authors about ORC being critical for their regulation seem premature without evidence that there is a direct mechanistic connection between ORC and the expression patterns observed rather than these changes being a general response to the altered cell cycle patterns observed when ORC is depleted.

Reviewer #3:

The authors present an interesting manuscript leading to four critical conclusions: 1. ORC proteins associate onto chromatin independently of their association as a holocomplex; 2. CDC6 and CDT1 are recruited to chromatin independently of ORC1 and ORC2; 3. enough MCM proteins are loaded to ensure a nearly undisrupted S-phase (albeit diminished cell proliferation) in the absence of ORC1 or ORC2; and 4. ORC proteins are important for the repression of Rb and PcG genes.

Using CRISPR-CAS9, neither ORC1 nor ORC2 deletions seemed to delay S-phase progression or affect the survival of the selected clones (passaged up to a year).

While ORC2-null cells modestly reduced the number of active origins of replication (~20% increase in distance between origins), ORC1-null cells did not show a loss of origin firing (perhaps even a slight increase). Unlike RNAi-based publications, ORC2 deletion did not lead to HP1 foci disruption or loss of chromatin condensation, but did delay mitosis and deregulate centrosomes.

The authors also present gene microarray and Gene Set Enrichment Analyses uncovering a global loss of silencing over genes regulated by Rb and PRC2 when either ORC1 or ORC2 are deleted.

While the claims are highly interesting, the data seems to scratch the surface when it comes to the relation between ORC, CDC6-CDT1 and MCM proteins. The conclusions that ORC proteins associate on chromatin without forming a complex, and that CDC6 and CDT1 deposit MCM proteins independently of ORC, nearly solely rely on a single western analysis of cells fractionated into soluble extracts and chromatin.

That the ORC proteins associate with chromatin independently of their association as a holocomplex can be further verified by IF, or by western analyses of the extracts separated by size (either through chromatography or on density gradients), for example. That CDC6 and CDT1 are recruited to chromatin independently of ORC is a strong claim and also needs further validation (IF analysis through cell division, etc.). Same goes for MCM deposition in the absence of a functional ORC complex (though that in itself could be a separate paper).

The manuscript should be strongly considered for publication, but only after the claims regarding ORC and CDC6-CDT1 association with chromatin in the absence of ORC are further investigated.

[Editors’ note: A previous version of this manuscript was rejected at revised stage, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "Human ORC is dispensable for DNA replication initiation, but essential for repression of retinoblastoma regulated genes." for consideration by eLife. Your article has been seen by the Reviewing Editor in consultation with one of the original reviewers and overseen by Kevin Struhl as the Senior Editor. The reviewer has opted to remain anonymous. Based on considerable discussion, we regret to inform you that your work will not be considered further for publication in eLife.

We recognize the importance and surprise to the field of the definitive demonstration that not all the ORC subunits are required for replication initiation or even cell viability. However, the concern expressed during the original review was whether ORC was dispensable (as implied in the original paper and would be a major shock) or whether individual subunits were dispensable. In response to the original reviews, the authors provided new data addressing the role of Orc5. Unfortunately, the results are not clear cut, and the authors simply state that they can't say that the other subunits are required or not. While not conclusive, the observed effects of Orc5 depletion as well as the failure to obtain knockouts of other Orc subunits suggest that a partial or altered ORC complex is necessary and sufficient for viability and replication initiation. If true, this result would be less interesting because many multiprotein complexes contain subunits that are not essential for the function of the complex. In any event, the limited information on how replication occurs in the absence of Orc1 or Orc2 renders this paper as an unexpected and interesting observation with little mechanistic understanding. In addition, the transcription data is very preliminary because there are many direct and indirect ways that gene expression changes can occur and this data is peripheral to the central point of the paper about the role of ORC in DNA replication. For these reasons, the work better suited for a more specialized journal.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Two subunits of human ORC are dispensable for DNA replication and proliferation" for further consideration at eLife. Your revised article has been favorably evaluated by Kevin Struhl (Senior and Reviewing editor) and one reviewer.

The manuscript has been improved but there is one remaining issue that needs to be addressed before acceptance. Specifically, the Orc5 data is interpreted in a way that masks the finding that Orc5 depletion is still important in the absence of Orc1 or Orc2 (last paragraph of the results). The current version concludes that Orc5 is no more important than what is observed when Orc1 and Orc2 is present and hat it does not become more important. The reviewer and I think that the correct conclusion is that Orc5 remains important even in the absence of the other factors, and this is further supported by the now unmentioned result that the authors have been unable to create viable deletions of other Orc subunits (recognizing that this could be a trivial result, but still in contrast to what is observe for Orc1 and Orc2). In fact, it would be useful for the paper to mention the failure to get viable deletions of other Orc subunits, of course with the above caveat. The latter interpretation supports the idea that at least one of the non-deleted subunits is still involved in replication (an important conclusion). If this issue is taken care of, the paper will be accepted.

https://doi.org/10.7554/eLife.19084.010

Author response

Summary:

All reviewers find the work potentially important, but to validate that ORC is not essential for DNA replication additional work is required as stated below. Additionally, the experiments suggesting that the ORC complex is important for expression of Rb- and Polycomb target genes is based on single experiments. We suggest that you expand on these experiments or resubmit a shorter version focusing exclusively on the DNA replication part.

The influence of ORC on RB can be explained by Stillman’s recent paper in eLife (Hossain and Stillman, 2016. Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription. eLife, 5, pp. 10.7554/eLife.12785), which shows physical interaction between ORC1 and RB. So we have retained that result and cite the Stillman paper as a possible mechanism. We have removed the ORC control of PRC2 regulation, as suggested by you.

Essential revisions:

We all agree that the statement based on the deletion of ORC1 and ORC2, while suggestive of the conclusion, does not provide a definitive answer for ORC-independent DNA replication. We would like to see a deletion or knock down of ORC5.

We have been unable to delete both alleles of ORC4, 5, or 6. Since this is a negative result, we cannot say whether it says that these genes are essential, or whether we will eventually get a deletion after more attempts.

siRNA of ORC5 decreased BrdU incorporation (DNA synthesis) by 60% in WT cells, and only 30% in the ORC1-/- or ORC2-/- cells. Again we cannot conclude from this that ORC5 is non-essential because siRNA knockdown can still leave residual ORC5. This result has now been included in Figure 10 and discussed in the paper.

Please note that our paper is very careful to say that replication is independent of complete ORC (the origin recognition complex composed of 6 subunits). We are not (yet) claiming that all ORC subunits are non-essential for replication. This is now better stated in the paper.

Reviewer #1:

In this manuscript, the authors aim to address the roles of ORC2 and ORC1 proteins in replication by knocking out these proteins in HCT116 and other cell lines. They observe no drastic effects on DNA replication, cell cycle progression, origin number and distribution. They also find that CDC6 plays an important role in this context and that a large number of PRC2 and Rb -dependent genes are deregulated.

Major comment:

Although these findings are important, they do not offer much novel insights. The authors have previously shown a number of the current findings in a hypomorphic ORC2 cell line (JBC (10), 6253-6260). Also, that ORC has roles in gene silencing also has been shown (Genes & Dev (9), 911-924 / Cell (91), 311-323). Additional data that provide some novel mechanistic insight into why ORC is dispensable for replication or what kind of role it plays in transcriptional regulation of PRC2 and Rb regulated genes are necessary to justify publication of this manuscript in eLife.

We respectfully point out that our paper is the first to show that ORC has a role in gene silencing independent of its role in DNA replication. Jasper Rine’s 1995 paper was with ORC in S. cerevisiae. Michael Botchan’s 1997 paper was with ORC in D. melanogaster. In both the organisms ORC is also essential for replication initiation and so they were unable to conclude that ORC’s function in replication and gene regulation are independent activities. We show that the replication function and gene expression regulation functions of ORC are separable in mammalian cells. In addition, the positive results on transcription (that loss of ORC deregulates gene expression) provide a great positive control that attests to the successful depletion of functional ORC. This positive result is worth contrasting with the negative effects on DNA replication (that loss of ORC does not have much of an effect on DNA replication initiation).

We now refer to Stillman’s recent paper to cite a possible mechanism by which ORC could regulate RB (Hossain and Stillman, 2016. Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription. eLife, 5, pp. 10.7554/eLife.12785). ORC 1 protein reduction in the ORC1 KO or the ORC2 KO cells may explain the deregulation of RB regulated genes. We removed the transcriptional control of PRC2 regulation because we do not know the mechanism by which ORC regulates PRC2.

Reviewer #2:

The manuscript by Dutta and colleagues describes the analysis of deletions of ORC2 or ORC1, key subunits of the origin-recognition complex (ORC), in human cells. ORC is an essential replication protein in simple eukaryotes where origins of replication are defined by specific sequences. In contrast, higher eukaryotes lack a consensus sequence that defines origins and the molecular determinants of the sites of replication initiation in these organisms remain murky. The authors make the intriguing finding that deletions of either the ORC1 or the ORC2 gene are not lethal in human tissue culture cells. They go on to show that loading of the essential Mcm2-7 helicase occurs at reduced levels in these strains but residual loading is observed. Because the chromatin association of Cdc6 and Cdt1, two other helicase-loading proteins is unaffected, the authors suggest that continued association of these proteins mediates the residual Mcm2-7 loading. Intriguingly, depletion of Cdc6 reduced but did not eliminate replication in these cells (perhaps due to incomplete depletion).

The authors do a nice job of showing that deletions of ORC1 and ORC2 are viable and maintain relatively normal patterns of replication. The authors have also done well to show that their deletions are complete and there is not large amounts the proteins retained in the targeted cells. What is less clear is the method by which the residual Mcm2-7 is being loaded. Clearly Cdc6 is still playing a role but whether the remaining ORC subunits are capable of assisting with this is not clear. It would significantly strengthen the paper to show that siRNA of other ORC subunits does not have the same effect as there is either substantial (ORC1 deletion) or residual (ORC2 deletion) binding of other ORC subunits in each case. Overall, this part of the paper convincingly shows that there are other pathways than the canonical ORC-dependent ones to initiate replication.

The siORC5 result is now included in Figure 10. siRNA of ORC5 decreased BrdU incorporation (DNA synthesis) by 60% in WT cells, and only 30% in the ORC1-/- or ORC2-/- cells. Again we cannot conclude from this that ORC5 is non-essential because siRNA knockdown can still leave residual ORC5. This result has now been included in Figure 10 and discussed in the paper.

Our results show that replication is independent of complete ORC (The origin recognition complex composed of 6 subunits). We are not claiming that all ORC subunits are non-essential.

The authors also observe that the loss of ORC causes changes in gene expression patterns that are similar to E2F1 over-expression strains, Rb knockdown strains and PRC2 subunit deletion strains. While these findings indicate that deletion of ORC subunits leads to changes in the patterns of gene expression, whether these changes are direct or indirect is unclear. The authors suggest that ORC is "critical for the regulation of these genes," however, they do not discuss the possibility that the changes in cell cycle progression as a possible reason for these changes. It would improve the paper to have a more complete discussion of the various reasons that these genes could be altered.

There is not much difference in cell cycle progression in the ORC KO cells based on FACS profile or S-phase progression. (Figure 2B, C, 7B, and C), therefore we do not think that a change in cell cycle progression is responsible for the gene expression change. In addition, we now cite Stillman’s recent paper in eLife for a possible explanation of how ORC could regulate RB targeted genes.

One other point that merits additional discussion is the selection that is going on as they grow up cells after deletion of ORC1 or ORC2. The authors point out that over time these cells are dividing faster which strongly suggests that there are additional genetic changes that alow improved cell growth. This means that the cells being analyzed are not simply ORC1-/- cells or ORC2-/- cells. It is important that the authors acknowledge this caveat and discuss what types of changes could be responsible for the improved growth.

We have acknowledged this caveat. We do not know what change (genetic, epigenetic, or post transcriptional) occurred in ORC1-/- cells as they were passaged. In the future, we plan to do to whole genome sequencing, RNA sequencing, and Mass spec analysis from ORC1 KO cells in early and late passage to determine the mechanism of this adaptation. This is now discussed.

Overall, the authors provide data that strongly suggests that there are non-canonical methods to initiate replication in the absence of ORC1 or ORC2. This is an important and surprising insight into the mechanism of initiation in mammalian cells and is consistent with previous data from this lab and in Drosophila that there could be ORC-independent mechanisms for replication initiation. It remains to be demonstrated whether the residual DNA replication observed is independent of the other ORC subunits (that is truly ORC- independent) or not (this would require double mutants in ORC – it would be a very nice addition if the authors showed that depletion/elimination of a second ORC subunit in the ORC1-/- or ORC2-/- cells did or did not further reduce replication). The gene expression data is relatively underdeveloped and the strong statements made by the authors about ORC being critical for their regulation seem premature without evidence that there is a direct mechanistic connection between ORC and the expression patterns observed rather than these changes being a general response to the altered cell cycle patterns observed when ORC is depleted.

Discussed above.

Reviewer #3:

[…]That the ORC proteins associate with chromatin independently of their association as a holocomplex can be further verified by IF, or by western analyses of the extracts separated by size (either through chromatography or on density gradients), for example. That CDC6 and CDT1 are recruited to chromatin independently of ORC is a strong claim and also needs further validation (IF analysis through cell division, etc.). Same goes for MCM deposition in the absence of a functional ORC complex (though that in itself could be a separate paper).

We have performed immunolfuorescence of MCM5 after extraction of nucleiplasmic proteins and confirmed the lower intensity of MCM5 staining in ORC1 or ORC2 KO cells. These data are now included as Figure 1—figure supplement 5.

We can co-IP ORC2 and ORC3 in the ORC1 -/- cells (data not shown), but neither this experiment nor the experiment the reviewer suggests addresses whether the chromatin association of MCM2-7 is effected by an ORC sub complex, or by individual ORC subunits. To test whether interactions between the residual ORC subunits (ORC subcomplex formation) is necessary for the residual ORC or MCM loading, we have to knock in mutations that disrupt interactions between the remaining ORC subunits and test the phenotype. As the reviewer suggests, that would clearly be a separate paper.

[Editors' note: the author responses to the re-review follow.]

We recognize the importance and surprise to the field of the definitive demonstration that not all the ORC subunits are required for replication initiation or even cell viability. However, the concern expressed during the original review was whether ORC was dispensable (as implied in the original paper and would be a major shock) or whether individual subunits were dispensable. In response to the original reviews, the authors provided new data addressing the role of Orc5. Unfortunately, the results are not clear cut, and the authors simply state that they can't say that the other subunits are required or not. While not conclusive, the observed effects of Orc5 depletion as well as the failure to obtain knockouts of other Orc subunits suggest that a partial or altered ORC complex is necessary and sufficient for viability and replication initiation. If true, this result would be less interesting because many multiprotein complexes contain subunits that are not essential for the function of the complex. In any event, the limited information on how replication occurs in the absence of Orc1 or Orc2 renders this paper as an unexpected and interesting observation with little mechanistic understanding. In addition, the transcription data is very preliminary because there are many direct and indirect ways that gene expression changes can occur and this data is peripheral to the central point of the paper about the role of ORC in DNA replication. For these reasons, the work better suited for a more specialized journal.

Thank you for the decision letter and for talking with me about the issues raised by the reviewers. I appreciate the fact that the reviewers believe that the cells are viable and replicating in the absence of Orc2 or Orc1, but are concerned that we have not ruled out that a partial or altered subcomplex of the residual subunits is somehow eking out replication. This is my appeal asking for a re-consideration. If you agree, we can incorporate these points in the discussion.

1) I agree that we cannot rule out the possibility that a partial or altered subcomplex of the residual ORC subunits (comprising perhaps Orc3-4-5-6) carries out the function of the six- subunit ORC. However, I believe that such a possibility still makes us reconsider how ORC functions. Jim Berger’s beautiful structure of ORC (1) makes the following suggestions:

a) Orc2-3-5-4-1 are arranged in a gapped ring (in that order) with a central channel of 20A that is wide enough to surround a DNA double-helix, and that later in licensing, Cdc6 slips into the gap between Orc2 and Orc1 to close the gap (The schematic from their paper shows the ring of the Winged-Helix domains of ORC subunits). The ORC-Cdc6 ring is proposed to interact with the MCM2-7 ring end-on-end during the loading of MCM2-7. Loss of Orc2 or of Orc1 makes it difficult for the remaining subunits to form a ring large enough (i) to surround a DNA double-helix in the same manner as wild type ORC or (ii) to interact with the MCM2-7 ring end-on-end.

b)The structure of ORC has two tiers of rings offset with respect to each other, a tier of Winged-Helix (WH) domains and a tier of AAA+ domains. The WH domain of Orc1 interacts with the AAA+ domain of Orc4, and the AAA+ like domain of Orc2 interacts with the WH domain of Orc3. One could suggest that another cellular molecule substitutes for the missing Orc subunit in the two-tiered ring. However, the alternate molecule that replaces Orc1 (in the Orc1-/- cells) or Orc2 (in the Orc2-/- cells) not only has to have a WH domain and an AAA+ domain in a similar configuration as Orc subunits, but also has to retain the specific interactions that allow the Orc1WH-Orc4AAA or the Orc2AAA-Orc3WH interactions mentioned above.

c) Cdc6 is proposed to slip into the gap between Orc2 and Orc1 to close the ring and lock the DNA in place. Deletion of Orc2 or Orc1 creates problems for that model, because Cdc6 interacts with the Orc2 and Orc1 subunits of the open ring to close the latter.

d) The AAA+ domain of Orc1 blocked the central channel in the crystal structure and it was proposed that this is an auto-inhibited form of ORC that will be activated at the correct time in the cell-cycle to allow the DNA to slip into place in the central channel. Such autoinhibition is clearly not necessary because replication can proceed independent of Orc1.

2) Biochemical analyses of human ORC formation carried out 15 years back in my lab and that of others (2-4) proposed that Orc2 together with Orc3 is the core of the complex (the Orc2-3 dimer forms easily without other subunits). The Orc2-3 complex can associate with Orc5 but not Orc4. The Orc2-3-5 complex brings in Orc4, and finally the Orc2-3-5-4 complex associates with Orc1. This order of subunits entering the complex (Orc2-3-5-4-1) exactly recapitulates the order of the subunits around the ring in the Berger structure. It is hard to imagine how a residual complex can be formed in the absence of Orc2.

3) Human Orc1 and Orc4 are the only subunits that have intact Walker A and B motifs. The other subunits (Orc2, 3 and 5) have AAA+-like domains but not intact Walker A and B motifs. Multiple groups have shown that the ATPase activity of ORC (in S. cerevisiae, in D. melanogaster and in H. sapiens) depends exclusively on the Walker A and B motifs of the Orc1 subunit, and that this ATP binding and hydrolysis activity is essential for ORC function (5-7). For example, Orc1 (and the Walker A motif of Orc1) is essential for human ORC to bind to chromatin and to support DNA replication in Xenopus egg extracts (6). Thus has risen the model that the ATP binding and hydrolysis by the Orc1 subunit of ORC is critical for replication licensing. This model has to be reconsidered now that we show that cells are viable without Orc1, even if an altered or partial ORC is initiating replication. If an alternate cellular molecule substitutes for Orc1 in this critical function, it is most likely Cdc6. This is why we like the result that the Cdc6 gene becomes essential for cell proliferation in the cells deleted for Orc1 (or Orc2).

4) The BAH (Bromo-Adjacent-Homology) domain of Orc1 has been proposed to interact with nucleosomes, and this interaction deemed to be essential for origin selection and replication (8,9). A mutation in the BAH domain of Orc1 leads to Meier-Gorlin syndrome. Even if Cdc6 were to substitute for Orc1, Cdc6 does not have a BAH domain. Thus the importance of ORC- nucleosome interaction in origin specification has to be reconsidered.

5) When we have such a surprising result on the replication front, it is comforting to know that the at least another proposed function of ORC (regulation of gene expression) is evident in the Orc1-/- or Orc2-/- cells. The robust and reproducible changes in the gene expression program assure the reader that we have affected something in the cell upon deletion of Orc1 or Orc2. That is why we would like to keep it in the paper, but will delete it if you make that a pre- condition for accepting the paper in eLife. We think the regulation of Rb/E2F genes by ORC is explained by what Stillman has just published in 2016 in eLife (10).

6) There is a paper in EMBO J. suggesting that Orc2 is essential for properly condensed mitotic chromosomes and chromosome congression in the mitotic plate (11). This is why we thought it useful to show that Orc2 deletion had no effect on these phenotypes. Also, if there is residual DNA damage in cells replicating in the absence of Orc2, we would expect a severe block to entry into M phase (because of activation of the S to M checkpoint). This is why we examined entry and passage through mitosis of these cells. We can take out all this data if you make it a precondition for acceptance in eLife.

7) There is a paper in Science suggesting that Orc1 knockdown leads to 25-40% of cells having multiple centrosomes, compared to 2.5% of control cells (12). (From the abstract: “We report a new role for the Orc1 protein, a subunit of the Origin Recognition Complex (ORC) that is a key component of the DNA replication licensing machinery in controlling centriole and centrosome copy number in human cells, independent of its role in DNA replication.”) This is why we show that Orc2-/- cells barely increase centrosome number compared to WT cells (from 10% to 15%). We have similar data for Orc1-/- cells but did not include it in the paper. We can leave (or add to) this data in the paper, but can take it out if you make it a precondition for acceptance in eLife.

References:

1. Bleichert F, Botchan MR, Berger JM. Crystal structure of the eukaryotic origin recognition complex. Nature. 2015;519(7543):321-6. doi: 10.1038/nature14239. PubMed PMID: 25762138; PMCID: PMC4368505.

2. Dhar SK, Delmolino L, Dutta A. Architecture of the human origin recognition complex. J Biol Chem. 2001;276(31):29067-71. PubMed PMID: 11395502.

3. Vashee S, Simancek P, Challberg MD, Kelly TJ. Assembly of the human origin recognition complex. J Biol Chem. 2001;276(28):26666-73. doi: 10.1074/jbc.M102493200. PubMed PMID: 11323433.

4. Siddiqui K, Stillman B. ATP-dependent assembly of the human origin recognition complex. J Biol Chem. 2007;282(44):32370-83. doi: 10.1074/jbc.M705905200. PubMed PMID: 17716973.

5. Chesnokov I, Remus D, Botchan M. Functional analysis of mutant and wild-type Drosophila origin recognition complex. Proc Natl Acad Sci U S A. 2001;98(21):11997-2002. doi: 10.1073/pnas.211342798. PubMed PMID: 11593009; PMCID: PMC59756.

6. Giordano-Coltart J, Ying CY, Gautier J, Hurwitz J. Studies of the properties of human origin recognition complex and its Walker A motif mutants. Proc Natl Acad Sci U S A. 2005;102(1):69-74. doi: 10.1073/pnas.0408690102. PubMed PMID: 15618391; PMCID: PMC544074.

7. Klemm RD, Austin RJ, Bell SP. Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. Cell. 1997;88(4):493-502. PubMed PMID: 9038340.

8. Kuo AJ, Song J, Cheung P, Ishibe-Murakami S, Yamazoe S, Chen JK, Patel DJ, Gozani O. The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome. Nature. 2012;484(7392):115-9. doi: 10.1038/nature10956. PubMed PMID: 22398447; PMCID: PMC3321094.

9. Muller P, Park S, Shor E, Huebert DJ, Warren CL, Ansari AZ, Weinreich M, Eaton ML, MacAlpine DM, Fox CA. The conserved bromo-adjacent homology domain of yeast Orc1 functions in the selection of DNA replication origins within chromatin. Genes Dev. 2010;24(13):1418-33. doi: 10.1101/gad.1906410. PubMed PMID: 20595233; PMCID: PMC2895200.

10. Hossain M, Stillman B. Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription. ELife. 2016;5. doi: 10.7554/eLife.12785. PubMed PMID: 27458800; PMCID: PMC4987141.

11. Prasanth SG, Prasanth KV, Siddiqui K, Spector DL, Stillman B. Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. EMBO J. 2004;23(13):2651-63. doi: 10.1038/sj.emboj.7600255. PubMed PMID: 15215892; PMCID: PMC449767.

12. Hemerly AS, Prasanth SG, Siddiqui K, Stillman B. Orc1 controls centriole and centrosome copy number in human cells. Science. 2009;323(5915):789-93. doi: 10.1126/science.1166745. PubMed PMID: 19197067; PMCID: PMC2653626.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for responding favorably to our appeal and allowing us to submit the paper as a Short Report. You make it clear that:

“It should focus on the key result (i.e. no transcriptional experiments) and a discussion of how the results are interpreted in terms of current knowledge. In particular, the discussion should explain how the results challenge the current view of ORC in terms of structural and biochemical knowledge, not a claim (not accepted by reviewers) that replication can occur without ORC.”

Below is a point-by-point dispensation of these issues.:

1) “No transcriptional experiments”: All results other than those pertaining to DNA replication have been removed.

2) “Not a claim (not accepted by reviewers) that replication can occur without ORC”: Done. We have even changed the title to say that “Two subunits of human ORC are dispensable…”.

3) “Focus on how the results challenge the current view of ORC”: We have done this. The Discussion is re-written to say that missing one subunit of a six-subunit ring that (a) encircles DNA and (b) interacts with MCM2-7 end-to-end, produces a spatial problem that must be somehow partially overcome in these cells. We also point out that the results show that all six subunits of ORC do not need to associate with chromatin in human cells as a holocomplex and that survival in the absence of ORC1 either suggests that the ATPase activity of ORC can be provided by ORC4 or CDC6, unlike the existing notion that ORC1 is the only subunit responsible for the ATPase activity of ORC.

https://doi.org/10.7554/eLife.19084.011

Article and author information

Author details

  1. Etsuko Shibata

    Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, United States
    Contribution
    ES, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Manjari Kiran

    Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, United States
    Contribution
    MK, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  3. Yoshiyuki Shibata

    Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, United States
    Contribution
    YS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  4. Samarendra Singh

    Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, United States
    Contribution
    SS, Acquisition of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  5. Shashi Kiran

    Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, United States
    Contribution
    SK, Acquisition of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  6. Anindya Dutta

    Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, United States
    Contribution
    AD, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    ad8q@eservices.virginia.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4319-0073

Funding

National Institutes of Health (CA060499)

  • Anindya Dutta

National Institutes of Health (CA166054)

  • Anindya Dutta

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dutta lab members for the useful discussion. This study is supported by R01 CA060499 and CA166054 (to AD).

Reviewing Editor

  1. Kevin Struhl, Harvard Medical School, United States

Publication history

  1. Received: June 23, 2016
  2. Accepted: December 1, 2016
  3. Accepted Manuscript published: December 1, 2016 (version 1)
  4. Version of Record published: January 19, 2017 (version 2)

Copyright

© 2016, Shibata et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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