RESUMO
This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.
Assuntos
Aminoglicosídeos , Antibacterianos/toxicidade , Núcleo Celular/metabolismo , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , DNA/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Zinostatina/toxicidade , Antibióticos Antineoplásicos/toxicidade , Western Blotting , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Sistema Livre de Células/efeitos dos fármacos , DNA/biossíntese , DNA Viral/antagonistas & inibidores , DNA Viral/biossíntese , Eletroforese em Gel de Poliacrilamida , Enedi-Inos , Humanos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Fosforilação/efeitos dos fármacos , Proteína de Replicação A , Vírus 40 dos Símios/genética , Solubilidade , Moldes Genéticos , Zinostatina/análogos & derivadosRESUMO
Human papillomaviral (HPV) origin-containing plasmids replicate efficiently in human 293 cells or cell extracts in the presence of HPV origin-recognition protein E2 and replication initiation protein E1, whereas cervical carcinoma-derived, HPV-18-positive HeLa cells or cell extracts support HPV DNA replication poorly. We recently showed that HPV-11 E1 interacts with cyclin/cyclin-dependent kinase (cdk) complexes through an RXL motif and is a substrate for these kinases. E1 mutations in this motif or in candidate cdk phosphorylation sites are impaired in replication, suggesting a role for cdks in HPV replication. We now demonstrate that one limiting activity in HeLa cells is cyclin E/CDK2. Purified cyclin E/CDK2 or cyclin E/CDK3 complex, but not other cdks, partially complemented HeLa cell extracts. Cyclin E/CDK2 expression vectors also enhanced transient HPV replication in HeLa cells. HeLa cell-derived HPV-18 E1 protein is truncated at the carboxyl terminus but can associate with cyclin E/CDK2. This truncated E1 was replication-incompetent and inhibited cell-free HPV replication. These results indicate that HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient HPV replication, most likely due to sequestration by the endogenous, defective HPV-18 E1 protein. Further analyses of the regulation of HPV E1 and HPV replication by cyclin E may shed light on the roles of cyclin E/CDK2 in cellular DNA replication.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina E/metabolismo , Papillomaviridae/metabolismo , Replicação Viral , Extratos Celulares , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Oncogênicas Virais/metabolismo , Plasmídeos , Proteínas Serina-Treonina Quinases , Origem de Replicação , Vírus 40 dos Símios/genética , Transfecção , Proteínas Virais/metabolismoRESUMO
The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesin-treated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Indóis , Vírus 40 dos Símios/genética , Afidicolina/farmacologia , Benzofuranos , Linhagem Celular Transformada , Cicloexenos , Duocarmicinas , Humanos , Cinética , Fosforilação , Proteína de Replicação A , Fase S , Vírus 40 dos Símios/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).
Assuntos
Alquilantes/farmacologia , Antivirais/biossíntese , Antivirais/farmacologia , Indóis/farmacologia , Vírus 40 dos Símios/efeitos dos fármacos , Ureia/análogos & derivados , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/fisiologia , Linhagem Celular , Sistema Livre de Células , Chlorocebus aethiops , Adutos de DNA/biossíntese , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , DNA Viral/biossíntese , DNA Viral/isolamento & purificação , Relação Dose-Resposta a Droga , Duocarmicinas , Eletroforese em Gel Bidimensional , Células HeLa , Temperatura Alta , Humanos , Vírus 40 dos Símios/química , Vírus 40 dos Símios/genética , Transativadores/biossíntese , Ureia/farmacologia , Replicação Viral/genéticaRESUMO
Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase alpha/primase (pol alpha/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol alpha/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2-dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.
Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , DNA Viral/biossíntese , Proteínas de Ligação a DNA/metabolismo , Papillomaviridae/genética , Papillomaviridae/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Ligação Competitiva , Domínio Catalítico , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , SpodopteraRESUMO
Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Origem de Replicação , Proteínas Virais/metabolismo , Proteínas de Transporte/metabolismo , Sistema Livre de Células , DNA Viral/biossíntese , Proteínas de Choque Térmico HSP40 , Humanos , Conformação de Ácido Nucleico , PapillomaviridaeRESUMO
The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.
Assuntos
Replicação do DNA , Proteínas Oncogênicas Virais/metabolismo , Proteínas Oncogênicas/metabolismo , Papillomaviridae/metabolismo , Replicação Viral , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Transformada , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Papillomaviridae/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Origem de Replicação , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.
Assuntos
Apolipoproteínas B/biossíntese , Proteínas de Transporte/metabolismo , Fígado/metabolismo , Edição de RNA , RNA Mensageiro/metabolismo , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/isolamento & purificação , Fracionamento Celular , Reagentes de Ligações Cruzadas , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Cinética , Neoplasias Hepáticas Experimentais , Metionina/metabolismo , Peso Molecular , RNA Neoplásico/metabolismo , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
We examined the functions of human papillomavirus type 11 (HPV-11) E1 and E2 proteins purified from Sf9 cells infected with recombinant baculoviruses in cell-free HPV-11 origin (ori) replication. The E1 protein binds specifically to a wild type but not to a mutated sequence in the ori spanning nucleotide position 1. It also has a relatively strong affinity for nonspecific DNA. A neutralizing antiserum directed against the amino-terminal one-third of the E1 protein totally abolishes initiation and elongation, suggesting that it functions as an initiator and a helicase at the replication fork. An antiserum against the carboxyl-terminal portion of E1 protein abolished replication only when added prior to initiation. Thus this portion of E1 is hidden in the replication complexes. The HPV-11 E2 protein appears not to be essential for elongation, but it must be present in the preinitiation complex for the E1 protein to recruit host DNA replication machinery to the ori. E2 antiserum added after preincubation in the absence of the cell extracts totally abolished replication. An identical conclusion is also reached for the bovine papillomavirus type 1 E2 protein. Finally, the HPV-11 E2C protein lacking the transacting domain of the full-length E2 protein partially inhibits E2-dependent ori replication.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Papillomaviridae/metabolismo , Proteínas Virais/metabolismo , Animais , Baculoviridae/genética , Bovinos , Linhagem Celular , Sistema Livre de Células , DNA Viral/biossíntese , DNA Viral/química , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Humanos , Técnicas In Vitro , Metilação , Papillomaviridae/genética , Origem de Replicação , Spodoptera , Proteínas Virais/genética , Replicação ViralRESUMO
We have established the first homologous cell-free DNA replication system for a papillomavirus. The replication of the human papillomavirus type 11 (HPV-11) origin was achieved by using human 293 cell extracts supplemented with the HPV-11 E1 and E2 proteins purified from insect cells infected with recombinant baculoviruses. Efficient replication depends on the HPV-11 origin, the HPV-11 E1 and E2 proteins, as well as human DNA polymerase alpha, delta, replication protein A, topoisomerase I, and topoisomerase II. High concentrations of E1 protein also promoted a low level of origin-independent replication which was suppressed by the addition of the E2 protein, whereas E2 protein stimulated origin-dependent replication. We also show that an intact E2 protein binding site was absolutely necessary for origin activity, as a strong HPV-11 origin was rendered inactive when one half-site of each of the three E2 binding sites was mutated. In contrast, there was only a relatively small reduction in this mutant origin activity when the cell extracts were supplemented with the bovine papillomavirus type 1 (BPV-1) proteins. These results suggest that the HPV-11 E2 protein plays a primary role in HPV origin recognition. Furthermore, unlike transient replication in which HPV-11 and BPV-1 viral proteins promote efficient replication of homologous and heterologous origins, efficient cell-free replication took place only with the homologous combinations.
Assuntos
Replicação do DNA , DNA Viral/biossíntese , Proteínas de Ligação a DNA/fisiologia , Papillomaviridae/genética , Proteínas Virais/fisiologia , Anticorpos , Sequência de Bases , Papillomavirus Bovino 1/genética , Linhagem Celular Transformada , Sistema Livre de Células , Epitopos , Humanos , Dados de Sequência Molecular , Origem de Replicação , Moldes GenéticosRESUMO
A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.