RESUMO
Eukaryotic DNA is organized into domains or loops generated by the attachment of chromatin fibers to the nuclear matrix via specific regions called scaffold or matrix attachment regions. The role of these regions in DNA replication is currently under investigation since they have been found in close association with origins of replication. Also, viral DNA sequences, containing the origins of replication, have been found attached to the nuclear matrix. To investigate the functional role of this binding we have studied, in Raji cells, the interaction between Epstein-Barr virus (EBV) origins of replication and the nuclear matrix in relation to the viral cycle of infection. We report here that both the latent (ori P) and the lytic (ori Lyt) EBV origins of replication are attached to the nuclear matrix, the first during the latent cycle of infection and the second after induction of the lytic cycle. These findings suggest that the binding of the origins of replication with the nuclear matrix modulates viral replication and expression in the two different phases of infection.
Assuntos
Infecções por Herpesviridae/genética , Herpesvirus Humano 4/genética , Origem de Replicação/genética , Infecções Tumorais por Vírus/genética , Latência Viral/genética , Mapeamento Cromossômico , DNA Viral/genética , DNA Viral/metabolismo , Genes Virais/genética , Herpesvirus Humano 4/fisiologia , Humanos , Matriz Nuclear/genética , Matriz Nuclear/metabolismo , Origem de Replicação/fisiologia , Células Tumorais Cultivadas , Ativação Viral/genética , Ativação Viral/fisiologiaRESUMO
Protein ERp60, previously found in the internal nuclear matrix in chicken liver nuclei, is a member of the protein disulfide isomerase family. It binds DNA and double helical polynucleotides in vitro with a preferential recognition toward the matrix-associated regions of DNA and poly(dA) x poly(dT), and its binding is inhibited by distamycin. ERp60 can be cross-linked chemically to DNA in the intact nuclei, suggesting that its association with DNA is present in vivo. As a whole, these results indicate that ERp60 is a component of the subset of nuclear matrix proteins that are responsible for the attachment of DNA to the nuclear matrix and for the formation of DNA loops. A distinctive feature of this protein, which has two thioredoxin-like sites, is that its affinity to poly(dA) x poly(dT) is strongly dependent on its redox state. Only its oxidized form, in fact, does it bind poly(dA) x poly(dT). The hypothesis can be made that through the intervention of ERp60, the redox state of the nucleus influences the formation or the stability of some selected nuclear matrix-DNA interactions.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Isomerases/metabolismo , Matriz Nuclear/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Galinhas , Reagentes de Ligações Cruzadas/metabolismo , Distamicinas/farmacologia , Isoenzimas/metabolismo , Fígado/enzimologia , Proteínas Nucleares/metabolismo , Oxirredução , Poli dA-dT/metabolismo , Tiorredoxinas/químicaRESUMO
DNA-protein cross-linkages were produced in intact nuclei of chicken erythrocytes by the action of cis-diammine dichloroplatinum. The telomeric DNA-protein cross-linked complexes were then isolated by hybridization with a biotinylated oligonucleotide and selective binding on immobilized streptavidin. Two main nonhistone proteins were present in the purified complexes, migrating in SDS-gel electrophoresis with apparent molecular masses of 66 and 58 kDa, respectively. Although the identity of these two proteins is still unknown, it is significant that two proteins with similar electrophoretic behavior have been described as constituents of the human telomeric complexes. This procedure could also be applied to the isolation of DNA-protein cross-linked complexes containing any chosen DNA sequence.
Assuntos
Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Eritrócitos/metabolismo , Telômero/metabolismo , Animais , Sequência de Bases , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Núcleo Celular/efeitos dos fármacos , Galinhas , Proteínas Cromossômicas não Histona/isolamento & purificação , Cisplatino/farmacologia , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eritrócitos/efeitos dos fármacos , Oligonucleotídeos , Telômero/efeitos dos fármacosRESUMO
DNA-protein cross-linkages were performed in intact undifferentiated and differentiated-HL60 cells by the action of cis-diammine dichloroplatinum. Total nuclear matrix proteins and DNA cross-linked nuclear matrix proteins were resolved by two-dimensional gel electrophoresis. The comparison of the electrophoretic patterns allowed the identification of a set of differentiation-induced nuclear matrix proteins cross-linked to DNA. One of these proteins binds cloned histone SAR sequences. Our results outline an experimental strategy for isolating and characterizing nuclear matrix components that may play a fundamental role in the overall control and coordination of gene expression during differentiation.
