ORC1 Research Articles

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1-6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2-7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9-mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2-7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2-7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2-7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2-7 to origins independent of ORC.

Chemical shifts of the WH domain of human ORC2

Chemical shifts of the WH domain of human ORC2

Human plasma complement C3 is independently associated with coronary heart disease, but only in heavy smokers (the CODAM study)

BackgroundComplement C3 is an emerging risk factor for coronary heart disease (CHD) and is particularly increased in the metabolic syndrome. A direct effect of smoking on structure and function of complement C3 has been suggested. HypothesisSmoking behavior may affect the cardiovascular risk that is associated with plasma complement C3. MethodsThe association between plasma C3 and CHD was studied in the CODAM (Cohort on Diabetes and Atherosclerosis Maastricht) study population (n=562, 61% male) with examination of effect modification by smoking. ResultsThe overall prevalence of CHD was 23.3%. Higher plasma C3 levels were associated with a higher CHD prevalence, and there was a significant interaction with heavy smoking (p=0.01). In never & light smokers, the univariate OR for CHD per 1s.d. (0.33g/L) increase in C3 was 1.09 [95% confidence interval (CI) 0.85–1.41] (p=0.505) whereas in heavy smokers it was 2.05 [1.43–2.93] (p<0.001). The association in the group of heavy smokers remained significant (OR 2.38 [1.54–3.68], p<0.001) after adjustment for traditional risk factors for cardiovascular disease and also after further adjustment for other cardiometabolic risk factors, i.e. the metabolic syndrome, CRP and insulin resistance (HOMA2IR) (OR C3 between 2.16 and 2.29, all p≤0.001). ConclusionHuman plasma complement C3 is associated with prevalent CHD, but only in heavy smokers, and this association is independent of important metabolic cardiovascular risk factors.

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats

Background The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet. Objective The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs. Methods Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by 3H thymidine ( 3H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically. Results Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in 3H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G 0/G 1 phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [ 3H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium. Conclusion ORC1 gene silencing causes rat VSMCs to enter a reversible G 0 quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.

Haplotype Mapping of a Diploid Non-Meiotic Organism Using Existing and Induced Aneuploidies

Haplotype maps (HapMaps) reveal underlying sequence variation and facilitate the study of recombination and genetic diversity. In general, HapMaps are produced by analysis of Single-Nucleotide Polymorphism (SNP) segregation in large numbers of meiotic progeny. Candida albicans, the most common human fungal pathogen, is an obligate diploid that does not appear to undergo meiosis. Thus, standard methods for haplotype mapping cannot be used. We exploited naturally occurring aneuploid strains to determine the haplotypes of the eight chromosome pairs in the C. albicans laboratory strain SC5314 and in a clinical isolate. Comparison of the maps revealed that the clinical strain had undergone a significant amount of genome rearrangement, consisting primarily of crossover or gene conversion recombination events. SNP map haplotyping revealed that insertion and activation of the UAU1 cassette in essential and non-essential genes can result in whole chromosome aneuploidy. UAU1 is often used to construct homozygous deletions of targeted genes in C. albicans; the exact mechanism (trisomy followed by chromosome loss versus gene conversion) has not been determined. UAU1 insertion into the essential ORC1 gene resulted in a large proportion of trisomic strains, while gene conversion events predominated when UAU1 was inserted into the non-essential LRO1 gene. Therefore, induced aneuploidies can be used to generate HapMaps, which are essential for analyzing genome alterations and mitotic recombination events in this clonal organism.

DDE BURDEN AND BREAST CANCER RISK

The exposure to DDT in relation to breast cancer (BC) risk received increasing attention since the beginning of the 90's. Contradicting results are published regarding the supposed relationship between body burden levels of p,p'-DDE -the main DDT metabolite- and BC. In this paper we argue that such conflict might be explained by methodological differences crosswise those studies. To such aim a meta-analysis of 22 articles was performed using the DerSimonian and Laird method for random effects models. The Q-statistic was used to identify heterogencity in the outcome variable across different studies. The gradient of p,p'-DDE exposure in epidemiological studies was homogenized to ng/g. The potential for publication bias was examined with the Begg's test and methodological features of studies are then discussed. The summary odds ratio for selected studies was 0.97 (95% CI, 0.87–1.09) in which the gradient of exposure ranged from 84.37 to 12,948 ng/g. No overall heterogeneity in the OR (c2 = 27.93; df = 23; p = 0.218) was observed. Neither the design of the study, nor the lack for breastfeeding control or the type of biological specimen where p,p'-DDE levels were measured, were causes of heterogeneity throughout the studies. Evidence of publication bias was not found (p = 0.253). These results do not lend support to the idea that a relationship between p,p'-DDE and BC risk could exist. Nevertheless, the exposure to DDT during critical periods of human development, from conception to adolescence, and individual variations in the metabolizing enzymes of DDT or its derivatives are still questions to be researched in regards to adult BC.

Mouse homolog of the yeast origin recognition complex subunit ORC1 and chromosomal localization of the cognate mouse gene Orc1.

ORC1 encodes a subunit of the eukaryotic origin recognition complex in yeast, which has been shown to play a crucial role in chromosomal DNA replication and transcriptional silencing. We have identified a mouse protein closely related to Saccharomyces cerevisiae ORC1 as well as to the human, Xenopus and Drosophila homologs. MmORC1 has putative cyclin-dependent phosphorylation sites and a nucleotide triphosphate-binding motif. We have mapped the gene locus of the murine Orc1 gene to chromosome 4, band D. Conservation of structures among members of the ORC1-related proteins suggests that these proteins play a key role in the intiation of DNA replication in all eukaryotes.

Isolation of Human and Fission Yeast Homologues of the Budding Yeast Origin Recognition Complex Subunit ORC5: Human Homologue (ORC5L) Maps to 7q22

Orc5p is a subunit of the origin recognition complex in the budding yeastSaccharomyces cerevisiae,which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing. We have cloned cDNAs from both human and fission yeastSchizosaccharomyces pombethat encode proteins homologous to the budding yeast andDrosophilaOrc5p. Human Orc5p showed 35.1, 22.3, and 19.4% identity to theDrosophila, S. pombe,andS. cerevisiaeOrc5p, respectively. We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescencein situhybridization. We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1. We have identified theS. pombeORC5 gene and located it in a cosmid mapped on chromosome II.

Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor.

The initiation of DNA replication in Saccharomyces cerevisiae requires the action of a multisubunit complex of six proteins known as the origin recognition complex (ORC). The identification of higher eukaryotic homologs of several ORC components suggests a universal role for this complex in DNA replication. We now demonstrate that the expression of one of these homologs is regulated by cell proliferation. Expression of the human Orc1 gene (HsOrc1) is low in quiescent cells, and it is then dramatically induced upon stimulation of cell growth. In contrast, expression of the HsOrc2 gene does not appear to be similarly regulated. We have isolated the promoter that regulates HsOrc1 transcription, and we show that the promoter confers cell growth-dependent expression. We also demonstrate that the cell growth control is largely the consequence of E2F-dependent negative transcription control in quiescent cells. Activation of HsOrc1 transcription following growth stimulation requires G1 cyclin-dependent kinase activity, and forced E2F1 expression can bypass this requirement. These results thus provide a direct link between the initiation of DNA replication and the cell growth regulatory pathway involving G1 cyclin-dependent kinases, the Rb tumor suppressor, and E2F.

The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase.

In a screen for new cell-cycle genes in Schizosaccharomyces pombe we have isolated cdc30, which is identical to orp1, a putative homolog of the Saccharomyces cerevisiae ORC1 gene. Analysis of the temperature-sensitive orp1-4 and the orp1(delta) mutants indicates that orp1 is required at the onset of S phase for an early step of DNA replication. Orp1p is found in the nucleus and is present at a constant level throughout the cell cycle. Genetic interactions occur between orp1 and cdc18 and cdc21 (an MCM homolog). Orp1p forms protein complexes with both cdc18p and cdc21p in vivo, suggesting that interactions between these proteins and ORC are important for controlling the initiation of DNA replication at the onset of S phase. The orp1 gene is also required for the control that prevents entry into mitosis in the absence of DNA replication, suggesting a role for ORC in this checkpoint pathway.

Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae.

A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC.

Complement bridges between cells analysis of a possible cell-cell interaction mechanism.

Different leukocytes (Raji, Daudi, Rael lymphoid cells; human peripheral blood lymphocytes, and guinea pig granulocytes), which had been coated with C3 by incubation of 37 degrees C for 20 min in a C3 solution, were demonstrated to form rosettes with erythrocytes coated with complement components (EAC142). The percentage of rosettes was dependent of the amount of C3 present on the cells. Loading of the lymphoid cells with C3 was a time- and temperature-dependent process. C3b was unable to serve the same purposes, although C3 and C3b occupied the C3 receptors on the lymphoid cells to a comparable degree. C3 functions in a similar manner. The C42 enzyme can be replaced by trypsin, so that bridging units may consist of C3 + C42, C5 + C42 OR C3 + trypsin, and C5 + trypsin. Bridging units can be constructed also from C4 + C1. It is suggested that enzymes on one cell liberate labile binding groups of complement components on adjacent cells, thus inducing coupling of the two cells. The possibility is raised that this type of cell interlinkage may play a role in vivo, since there is accumulating evidence that complement components are expressed in the plasma membrane of different cells.