RESUMO
Platinum-based drugs such as cisplatin (CP) are the first-line chemotherapy for non-small-cell lung carcinoma (NSCLC). Unfortunately, NSCLC has a low response rate to CP and acquired resistance always occurs. Histone methylation regulates chromatin structure and is implicated in DNA repair. We hypothesize histone methylation regulators are involved in CP resistance. We therefore screened gene expression of known histone methyltransferases and demethylases in three NSCLC cell lines with or without acquired resistance to CP. JMJD2s are a family of histone demethylases that remove tri-methyl groups from H3K9 and H3K36. We found expression of several JMJD2 family genes upregulated in CP-resistant cells, with JMJD2B expression being upregulated in all three CP-resistant NSCLC cell lines. Further analysis showed increased JMJD2 protein expression coincided with decreased H3K9me3 and H3K36me3. Chemical inhibitors of JMJD2-family proteins increased H3K9me3 and H3K36me3 levels and sensitized resistant cells to CP. Mechanistic studies showed that JMJD2 inhibition decreased chromatin association of ATR and Chk1 and inhibited the ATR-Chk1 replication checkpoint. Our results reveal that JMJD2 demethylases are potential therapeutic targets to overcome CP resistance in NSCLC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/patologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Neoplasias Pulmonares/metabolismo , MetilaçãoRESUMO
MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial-mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Instabilidade Cromossômica/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/genética , Citoesqueleto de Actina/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Dano ao DNA/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
Somatic human cells contain thousands of copies of mitochondrial DNA (mtDNA). In eukaryotes, natural transfer of mtDNA into the nucleus generates nuclear mitochondrial DNA (NUMT) copies. We name this phenomenon as "numtogenesis". Numtogenesis is a well-established evolutionary process reported in various sequenced eukaryotic genomes. We have established a molecular tool to rapidly detect and analyze NUMT insertions in whole genomes. To date, NUMT analyses depend on deep genome sequencing combined with comprehensive computational analyses of the whole genome. This is time consuming, cumbersome and cost prohibitive. Further, most laboratories cannot accomplish such analyses due to limited skills. We report the development of single-molecule mtFIBER FISH (fluorescence in situ hybridization) to study numtogenesis. The development of mtFIBER FISH should aid in establishing a role for numtogenesis in cancers and other human diseases. This novel technique should help distinguish and monitor cancer stages and progression, aid in elucidation of basic mechanisms underlying tumorigenesis and facilitate analyses of processes related to early detection of cancer, screening and/or cancer risk assessment.
Assuntos
Núcleo Celular/metabolismo , DNA Mitocondrial/metabolismo , Hibridização in Situ Fluorescente/métodos , Transporte Biológico , Linhagem Celular Tumoral , Núcleo Celular/genética , HumanosRESUMO
The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.
Assuntos
Melanócitos/citologia , Melanócitos/efeitos da radiação , Melanoma/patologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Raios Ultravioleta , Biomarcadores/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/metabolismo , Diploide , Relação Dose-Resposta à Radiação , Fibroblastos/efeitos da radiação , Humanos , Melaninas/metabolismo , Fosforilação/efeitos da radiação , Proteínas Quinases/metabolismo , Dímeros de Pirimidina/metabolismoRESUMO
SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function.
Assuntos
Dano ao DNA , DNA Helicases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , DNA Helicases/deficiência , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas Nucleares/deficiência , RNA Interferente Pequeno/genética , Fatores de Transcrição/deficiênciaRESUMO
We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m(-2) UVC in early, mid- and late-S phase. The endpoints measured were the following: (1) radio-resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC-induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10-fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra-S checkpoint activated by UVC-induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.
Assuntos
Dano ao DNA , Diploide , Fase S , Replicação do DNA , Fibroblastos/citologia , HumanosRESUMO
Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme in mammalian cells. The enzyme synthesizes polymers of ADP-ribose from the coenzyme NAD(+) and plays multifaceted roles in cellular responses to genotoxic stress, including DNA repair. It had been shown that mouse fibroblasts treated with a DNA methylating agent in combination with a PARP inhibitor exhibit higher cytotoxicity than cells treated with methylating agent alone. This lethality of the PARP inhibitor is dependent on apurinic/apyrimidinic (AP) sites in the DNA and the presence of PARP-1. Here, we show that purified PARP-1 is capable of forming a DNA-protein cross-link (DPC) by covalently attaching to the AP site. This DPC formation is specific to the presence of the natural AP site in DNA and is accompanied by a single-strand DNA incision. Cellular studies confirm the formation of PARP-1 DPCs during alkylating agent-induced base excision repair (BER) and formation of DPCs is enhanced by a PARP inhibitor. Using an N-terminal and C-terminal truncated PARP-1 we show that a polypeptide fragment comprising the zinc 3 and BRCT sub-domains is sufficient for DPC formation. The covalent attachment of PARP-1 to AP site-containing DNA appears to be a suicidal event when BER is overwhelmed or disrupted.
Assuntos
Reparo do DNA , DNA/química , Poli(ADP-Ribose) Polimerases/química , Animais , Células Cultivadas , Cisteína , Humanos , Camundongos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/análise , Estrutura Terciária de ProteínaRESUMO
The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here, the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts. Specifically, the relationship between nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2 and ATR/Chk1 was examined. The results show increased phosphorylation of H2AX, ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA synthesis and frequency of replicon initiation events in DNA-combed fibres. Taken together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2 DNA damage response pathway.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Titânio/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células Cultivadas , Quinase do Ponto de Checagem 2/genética , Meios de Cultura , Dano ao DNA/fisiologia , Fibroblastos/fisiologia , Histonas/genética , Histonas/metabolismo , Humanos , Nanopartículas Metálicas/química , Microscopia Acústica , Fosforilação , Titânio/químicaRESUMO
Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects. Stable knockdown of CTC1 or STN1 increases the incidence of anaphase bridges and multi-telomeric signals, indicating genomic and telomeric instability. STN1 knockdown also delays replication through the telomere indicating a role in replication fork passage through this natural barrier. Furthermore, we find that STN1 plays a novel role in genome-wide replication restart after hydroxyurea (HU)-induced replication fork stalling. STN1 depletion leads to reduced EdU incorporation after HU release. However, most forks rapidly resume replication, indicating replisome integrity is largely intact and STN1 depletion has little effect on fork restart. Instead, STN1 depletion leads to a decrease in new origin firing. Our findings suggest that CST rescues stalled replication forks during conditions of replication stress, such as those found at natural replication barriers, likely by facilitating dormant origin firing.
Assuntos
Replicação do DNA , Proteínas de Ligação a Telômeros/genética , Telômero/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Humanos , Proteína 1 de Ligação a Repetições Teloméricas/genéticaRESUMO
In summary, recently developed technologies have begun to draw back the curtain of mystery that obscures some of the basic mechanisms of DNA replication at multiple levels. Studies using extended DNA and chromatin fiber techniques have proven valuable for identifying the location of origins of replication at specific genomic sites and determining their temporal order of replication, for identifying and quantifying sites of DNA damage and localizing chromatin proteins in relation to sites of DNA replication. The future potential of these methods include further discoveries in functional genomics and contributions to the elucidation of the histone code. Such studies could prove very valuable in studies of the mechanisms of cancer development, aging, and other processes of disordered genomic functioning.
Assuntos
Replicação do DNA , Fase S , Cromatina/metabolismo , Cromatina/ultraestrutura , DNA/química , DNA/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells. However, the neutralization of methoxyamine is required prior to usage. Here we developed a convenient, specific SSB assay using alkaline gel electrophoresis (AGE) coupled with a neutral O-hydroxylamine, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes abasic sites (AP sites) to prevent their alkaline incision while still allowing for strong alkaline DNA denaturation. DNA from DT40 and isogenic polymerase ß null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE (OTX-AGE) assay. Time-dependent increases in SSBs were detected in each cell line with more extensive SSB formation in the null cells. These findings were supported by an assay that indirectly detects SSBs through measuring NAD(P)H depletion. An ARP-slot blot assay demonstrated a significant time-dependent increase in AP sites in both cell lines by 1mM MMS compared to control. Furthermore, the Pol ß-null cells displayed greater AP site formation than the parental DT40 cells. OTX use represents a facile approach for assessing SSB formation, whose benefits can also be applied to other established SSB assays.
Assuntos
Dano ao DNA , Reparo do DNA , Hidroxilaminas/química , Alquilantes/farmacologia , Animais , Galinhas , DNA/metabolismo , Eletroforese , Concentração de Íons de Hidrogênio , Hidroxilamina/farmacologia , Metanossulfonato de Metila/farmacologia , Mutagênicos , NADP/química , Fatores de TempoRESUMO
For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers. The specific pattern of BRG1 occupancy suggests it does not participate in origin selection but is involved in the firing of origins and the process of replication elongation. This latter function is confirmed by the fact that Brg1 mutant mouse embryos and RNAi knockdown cells exhibit a 50% reduction in replication fork progression rates, which is associated with decreased cell proliferation. This novel function of BRG1 is consistent with its requirement during embryogenesis and its role as a tumor suppressor to maintain genome stability and prevent cancer.
Assuntos
DNA Helicases/fisiologia , Replicação do DNA , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proliferação de Células , Cromatina/química , DNA Helicases/análise , DNA Helicases/genética , Proteínas de Ligação a DNA/análise , Desenvolvimento Embrionário , Células Eritroides/metabolismo , Células HeLa , Humanos , Camundongos , Mutação , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Fenótipo , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
We investigated whether apurinic/apyrimidinic (AP/abasic) sites were more frequent in regions of DNA replication in cells and whether their number increased during oxidative stress. DNA fiber spreading and fluorescent immunostaining were used to detect areas of DNA replication and sites of AP lesions in extended DNA fibers. The distribution of AP sites was determined in DNA fibers from vertebrate cells maintained under normal culture conditions or stressed with exogenous H(2)O(2). AP lesions per unit length were enumerated in bulk DNA or at replication sites. The background density of AP sites in DNA fibers was 5.4 AP sites/10(6) nt, while newly replicated DNA contained 12.9 AP sites/10(6) nt. In cells exposed to 20 µM H(2)O(2), AP sites in newly replicated DNA increased to 20.8/10(6) nt. Determinations of AP site density in bulk DNA by fiber analysis or standard slot blot assays agreed to within 10%. Our findings show that the fiber assay not only accurately determines the frequency of AP sites but also shows their distribution. They also reveal that there is increased susceptibility to oxidative damage in DNA regions undergoing replication, which may explain the previously observed clustering of AP sites.
Assuntos
Replicação do DNA , Animais , Linhagem Celular , Galinhas , Dano ao DNA , Microscopia de Fluorescência , Espécies Reativas de Oxigênio/metabolismoRESUMO
From in vitro studies, flap endonuclease 1 (FEN1) has been proposed to play a role in the long patch (LP) base excision repair (BER) subpathway. Yet the role of FEN1 in BER in the context of the living vertebrate cell has not been thoroughly explored. In the present study, we cloned a DT40 chicken cell line with a deletion in the FEN1 gene and found that these FEN1-deficient cells exhibited hypersensitivity to H(2)O(2). This oxidant produces genotoxic lesions that are repaired by BER, suggesting that the cells have a deficiency in BER affecting survival. In experiments with extracts from the isogenic FEN1 null and wild-type cell lines, the LP-BER activity of FEN1 null cells was deficient, whereas repair by the single-nucleotide BER subpathway was normal. Other consequences of the FEN1 deficiency were also evaluated. These results illustrate that FEN1 plays a role in LP-BER in higher eukaryotes, presumably by processing the flap-containing intermediates of BER.
Assuntos
Reparo do DNA/genética , DNA/genética , Endonucleases Flap/genética , Estresse Oxidativo/genética , Animais , Linhagem Celular , Galinhas , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA/genética , Células Eucarióticas/metabolismo , Peróxido de Hidrogênio/toxicidade , Nucleotídeos/genética , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Vertebrados/genética , Vertebrados/metabolismoRESUMO
BACKGROUND: The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins. RESULTS: Using immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication. CONCLUSION: In agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.
RESUMO
A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability.
Assuntos
Replicação do DNA , Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular , DNA/biossíntese , DNA/química , Imunofluorescência , Geminina , Células HeLa , Humanos , Interferência de RNA , Fase S/genéticaRESUMO
Damaged DNA binding protein 1, DDB1, bridges an estimated 90 or more WD40 repeats (DDB1-binding WD40, or DWD proteins) to the CUL4-ROC1 catalytic core to constitute a potentially large number of E3 ligase complexes. Among these DWD proteins is the human immunodeficiency virus type 1 (HIV-1) Vpr-binding protein VprBP, whose cellular function has yet to be characterized but has recently been found to mediate Vpr-induced G(2) cell cycle arrest. We demonstrate here that VprBP binds stoichiometrically with DDB1 through its WD40 domain and through DDB1 to CUL4A, subunits of the COP9/signalsome, and DDA1. The steady-state level of VprBP remains constant during interphase and decreases during mitosis. VprBP binds to chromatin in a DDB1-independent and cell cycle-dependent manner, increasing from early S through G(2) before decreasing to undetectable levels in mitotic and G(1) cells. Silencing VprBP reduced the rate of DNA replication, blocked cells from progressing through the S phase, and inhibited proliferation. VprBP ablation in mice results in early embryonic lethality. Conditional deletion of the VprBP gene in mouse embryonic fibroblasts results in severely defective progression through S phase and subsequent apoptosis. Our studies identify a previously unknown function of VprBP in S-phase progression and suggest the possibility that HIV-1 Vpr may divert an ongoing chromosomal replication activity to facilitate viral replication.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/fisiologia , HIV-1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Transporte/genética , Ciclo Celular/fisiologia , Células Cultivadas , Cromatina/metabolismo , Proteínas Culina/genética , Proteínas de Ligação a DNA/genética , Feminino , HIV-1/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Proteínas Serina-Treonina Quinases , Interferência de RNA , Ubiquitina-Proteína Ligases/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoRESUMO
During the S phase of the cell cycle, the entire genome is replicated. There is a high level of orderliness to this process through the temporally and topologically coordinated activation of many replication origins situated along chromosomes. We investigated the program of replication from origins initiating in early S phase by labeling synchronized normal human fibroblasts (NHF1) with nucleotide analogs for various pulse times and measuring labeled tracks in combed DNA fibers. Our analysis showed that replication forks progress 9-35 kilobases from newly initiated origins, followed by a pause in synthesis before replication resumes. Pausing was not observed near origins that initiated in the middle of S phase. No evidence for pausing near origins was found at the beginning of the S phase in glioblastoma T98G cells. Treatment with the S phase checkpoint inhibitor caffeine abrogated pausing in NHF1 cells in early S phase. This suggests that pausing may comprise a novel aspect of the intra-S phase checkpoint pathway or a related new early S checkpoint. Further, it is possible that the loss of this regulatory process in cancer cells such as T98G could be a contributing factor in the genetic instability that typifies cancers.
Assuntos
Replicação do DNA/fisiologia , DNA/metabolismo , Origem de Replicação/fisiologia , Fase S/fisiologia , Análise de Variância , Cafeína/farmacologia , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Fibroblastos , Citometria de Fluxo , Humanos , Origem de Replicação/genéticaAssuntos
Transformação Celular Neoplásica/genética , Replicação do DNA/fisiologia , Neoplasias/genética , Fase S/genética , Animais , Carcinógenos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , Humanos , Neoplasias/induzido quimicamente , Neoplasias/etiologia , Neoplasias/patologiaRESUMO
UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.