RESUMO
The structure of chromatin regulates the genetic activity of the underlying DNA sequence. We report here that the protein encoded by the proto-oncogene DEK, which is involved in acute myelogenous leukemia, induces alterations of the superhelical density of DNA in chromatin. The change in topology is observed with chromatin but not with naked DNA and does not involve dissociation of core histones from chromatin. Moreover, these effects require histone H2A/H2B dimers in addition to histone H3/H4. We additionally tested whether the DEK protein affects DNA-utilizing processes and found that the DEK protein substantially reduces the replication efficiency of chromatin but not of naked DNA templates.
Assuntos
Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona , DNA/biossíntese , Histonas/metabolismo , Proteínas Oncogênicas/fisiologia , Western Blotting , DNA Super-Helicoidal , DNA Viral/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Conformação de Ácido Nucleico , Proteínas de Ligação a Poli-ADP-Ribose , Proto-Oncogene Mas , Proteínas Recombinantes/metabolismo , Vírus 40 dos Símios/genética , Transcrição GênicaRESUMO
DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro.
Assuntos
Cromatina/genética , Replicação do DNA , Drosophila/genética , Origem de Replicação/genética , Animais , Drosophila/embriologia , Histonas/metabolismo , Nucleossomos/metabolismo , Vírus 40 dos Símios/genéticaRESUMO
We have used the SV40 in vitro replication system to analyze the replication efficiencies of SV40 minichromosomes associated with normal or hyperacetylated histones. We found that elongation of replication occurs with higher efficiency in hyperacetylated minichromosomes in comparison with normal minichromosomes. Our results indicate that the movement of the replication machinery through nucleosomal DNA is facilitated by charge neutralization due to acetylation of the histone tails.
