Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin

A hypomorphic mutation made in the ORC2 gene of a human cancer cell line through homologous recombination decreased Orc2 protein levels by 90%. The G1 phase of the cell cycle was prolonged, but there was no effect on the utilization of either the c-Myc or beta-globin cellular origins of replication. Cells carrying this mutation failed to support the replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV), and this defect was rescued by reintroduction of Orc2. Orc2 specifically associates with oriP in cells, most likely through its interaction with EBNA1. Geminin, an inhibitor of the mammalian replication initiation complex, inhibits replication from oriP. Therefore, ORC and the human replication initiation apparatus is required for replication from a viral origin of replication.     

Comparative analysis of pre-replication complex proteins in transformed and normal cells

This study examines the abundance of the major protein constituents of the pre-replication complex (pre-RC), both genome-wide and in association with specific replication origins, namely the lamin B2, c-myc, 20mer1, and 20mer2 origins. Several pre-RC protein components, namely ORC1-6, Cdc6, Cdt1, MCM4, MCM7, as well as additional replication proteins, such as Ku70/86, 14-3-3, Cdc45, and PCNA, were comparatively and quantitatively analyzed in both transformed and normal cells. The results show that these proteins are overexpressed and more abundantly bound to chromatin in the transformed compared to normal cells. Interestingly, the 20mer1, 20mer2, and c-myc origins exhibited a two- to threefold greater origin activity and a two- to threefold greater in vivo association of the pre-RC proteins with these origins in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited both similar levels of activity and in vivo association of these pre-RC proteins in both cell types. Overall, the results indicate that cellular transformation is associated with an overexpression and increased chromatin association of the pre-RC proteins. This study is significant, because it represents the most systematic comprehensive analysis done to date, using multiple replication proteins and different replication origins in both normal and transformed cell lines.     

Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.     

Mutations to Ku reveal differences in human somatic cell lines

NHEJ (non-homologous end joining) is the predominant mechanism for repairing DNA double-stranded breaks in human cells. One essential NHEJ factor is the Ku heterodimer, which is composed of Ku70 and Ku86. Here we have generated heterozygous loss-of-function mutations for each of these genes in two different human somatic cell lines, HCT116 and NALM-6, using gene targeting. Previous work had suggested that phenotypic differences might exist between the genes and/or between the cell lines. By providing a side-by-each comparison of the four cell lines, we demonstrate that there are indeed subtle differences between loss-of-function mutations for Ku70 versus Ku86, which is accentuated by whether the mutations were derived in the HCT116 or NALM-6 genetic background. Overall, however, the phenotypes of the four lines are quite similar and they provide a compelling argument for the hypothesis that Ku loss-of-function mutations in human somatic cells result in demonstrable haploinsufficiencies. Collectively, these studies demonstrate the importance of proper biallelic expression of these genes for NHEJ and telomere maintenance and they provide insights into why these genes are uniquely essential for primates.     

Ku antigen,an origin-specific binding protein that associates with replication proteins,is required for mammalian DNA replication

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.     

Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Delta/-) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2.     

Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis

Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.     

Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts

DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80(-/-) cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and c-myc origins. This contrasts with previously published studies using Ku80-deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems.     

In Vivo Association of Ku with Mammalian Origins of DNA Replication

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.     

DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi’s sarcoma-associated herpesvirus latent replication

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi’s sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/−) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.     

Differential binding of replication proteins across the human c-myc replicator

The binding of the prereplication complex proteins Orc1, Orc2, Mcm3, Mcm7, and Cdc6 and the novel DNA unwinding element (DUE) binding protein DUE-B to the endogenous human c-myc replicator was studied by chromatin immunoprecipitation. In G(1)-arrested HeLa cells, Mcm3, Mcm7, and DUE-B were prominent near the DUE, while Orc1 and Orc2 were least abundant near the DUE and more abundant at flanking sites. Cdc6 binding mirrored that of Orc2 in G(1)-arrested cells but decreased in asynchronous or M-phase cells. Similarly, the signals from Orc1, Mcm3, and Mcm7 were at background levels in cells arrested in M phase, whereas Orc2 retained the distribution seen in G(1)-phase cells. Previously shown to cause histone hyperacetylation and delocalization of replication initiation, trichostatin A treatment of cells led to a parallel qualitative change in the distribution of Mcm3, but not Orc2, across the c-myc replicator. Orc2, Mcm3, and DUE-B were also bound at an ectopic c-myc replicator, where deletion of sequences essential for origin activity was associated with the loss of DUE-B binding or the alteration of chromatin structure and loss of Mcm3 binding. These results show that proteins implicated in replication initiation are selectively and differentially bound across the c-myc replicator, dependent on discrete structural elements in DNA or chromatin.     

Ku80 facilitates chromatin binding of the telomere binding protein,TRF2

The Ku70/80 heterodimer is central to non-homologous end joining repair of DNA double-strand breaks and the Ku80 gene appears to be essential for human but not rodent cell survival. The Ku70/80 heterodimer is located at telomeres but its precise function in telomere maintenance is not known. In order to examine the role of Ku80 fibroblast strain. Following targeted Ku80 knockdown, telomere defects are observed and the steady state levels of the TRF2 protein are reduced. Inhibitor studies indicate that this loss of TRF2 is mediated by the proteasome, and degradation of TRF2 following Ku depletion appears to involve a decrease in chromatin binding of TRF2, suggesting that the Ku heterodimer enhances TRF2 chromatin association and that non-chromatin bound TRF2 is targeted to the proteasome.     

Protein phosphatase 1 dephosphorylates Orc2

Phosphorylation of Thr¹¹⁶ and Thr²²⁶ on Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2-5) from human chromatin and replication origins. The phosphorylated Orc2 becomes dephosphorylated in the late M phase of the cell cycle. Here we show that protein phosphatase 1 (PP1) dephosphorylates Orc2. Dephosphorylation of Orc2 was accompanied by associating the dissociated Orc subunits with chromatin. Inhibitors of PP1 preferentially inhibited the dephosphorylation of Orc2. The overexpression of the α, β and γ PP1 isoforms decreased the amount of phosphorylated Orc2, and the depletion of these isoforms by RNA interference increased the amount of phosphorylated Orc2. These results suggest that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin.     

冬凌草甲素对人结肠癌细胞株HCT116生长的影响及其机制研究

目的：研究冬凌草甲素（ORI）对人结肠癌细胞株HCT116生长的影响及其可能机制。方法：以体外培养的HCT116细胞为研究对象,给予不同浓度（0、2.5、5、10、20μM）ORI处理HCT116细胞不同时间（0、24、48、72 h）,通过MTT法检测其对HCT116细胞增殖的影响,DAPI染色观察其对细胞核的形态的影响,western blot检测细胞内β-catenin、c-myc蛋白表达的变化。结果：1ORI可显著抑制HCT116细胞的增殖,且此作用随着浓度和作用时间的增加或延长而增强（P〈0.05）。2ORI处理HCT116细胞24小时后,细胞核固缩的百分率随药物作用浓度的增加而增加。35、10、20μM ORI处理HCT116细胞24小时后,细胞内的β-catenin、c-myc蛋白水平均显著下调,且随着ORI浓度的增加逐渐减少。结论：ORI能以浓度和时间依赖性的方式抑制HCT116细胞的增殖,其机制可能与Wnt/β-catenin信号通路有关。     

Ku80 facilitates chromatin binding of the telomere binding protein,TRF2

The Ku70/80 heterodimer is central to non-homologous end joining repair of DNA double-strand breaks and the Ku80 gene appears to be essential for human but not rodent cell survival. The Ku70/80 heterodimer is located at telomeres but its precise function in telomere maintenance is not known. In order to examine the role of Ku80 beyond DNA repair in more detail, we have taken a knockdown approach using a human fibroblast strain. Following targeted Ku80 knockdown, telomere defects are observed and the steady state levels of the TRF2 protein are reduced. Inhibitor studies indicate that this loss of TRF2 is mediated by the proteasome and degradation of TRF2 following Ku depletion appears to involve a decrease in chromatin binding of TRF2, suggesting that the Ku heterodimer enhances TRF2 chromatin association and that non-chromatin bound TRF2 is targeted to the proteasome.Key words: Ku80, TRF2, chromatin, telomere, fibroblast     

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.     

KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair

Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.     

The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin.     

Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop

The study investigated the molecular basis of resveratrol (RSV)-evoked apoptosis in four (Bax+/-, Bax-/-, p53+/+, and p53-/-) HCT116 colon cancer cell lines. RSV induced apoptosis in all the cells in a dose-dependent manner; however, Bax+/- and p53+/+ cells were more susceptible than their knockout counterparts (Bax-/- and p53-/-, respectively). Using Bax+/- cells as a model, proteomic analysis revealed four RSV-responsive events: fragmentation of lamin A/C protein; increase in concentration of a more basic isoelectric variant of the ribosomal protein P0; and decrease in concentration of dUTPase as well as stathmin 1. Lamin A cleavage in response to RSV treatment was confirmed using Western blot analysis. Caspase-6 was activated, which was evidenced by cleavage and accumulation in active form of caspase-6 as well as upregulation of the protease activity. RSV-elicited lamin A cleavage and apoptosis were entirely abrogated by the peptide inhibitors of caspase-6. Likewise, partial knockdown of caspase-6 expression using small interfering RNA resulted in significant inhibition of RSV-elicited lamin A cleavage and apoptosis. Furthermore, the lower apoptosis sensitivity of the knockout cells (Bax-/- and p53-/-) correlated with the relatively reduced processing of caspase-6 and lamin A cleavage. Taken together, these data highlight the critical role of caspase-6 and its cleavage of lamin A in apoptotic signaling triggered by RSV in the colon carcinoma cells, which can be activated in the absence of Bax or p53.