ABSTRACT
The initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg(2+) and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag-DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.
Subject(s)
Antigens, Viral, Tumor/genetics , DNA, Viral/genetics , Replication Origin/genetics , Simian virus 40/genetics , Antigens, Viral, Tumor/chemistry , Binding Sites/genetics , DNA Replication/genetics , DNA, Viral/chemistry , Protein BindingABSTRACT
Topoisomerases provide the unlinking activity necessary for replication fork movement during DNA replication. It is uncertain, however, whether topoisomerases are also required for the initiation of replication. To investigate this point, we have performed pulse-chase experiments with SV40 minichromosomes as template to distinguish between the initiation and the elongation of replication. Using an unfractionated cytosolic extract as a source of replication functions, we found that the addition of topoisomerases at the initiation step significantly increased the number of active chromatin templates, whereas addition of topoisomerases at the elongation step had only minor effects. Minichromosomes with an extended chromatin structure as well as protein-free DNA required less topoisomerase for effective replication initiation. We could exclude the possibility that topoisomerases enhance the origin binding of T antigen, the SV40 replication initiator, and propose instead that the arrangement of nucleosomes influences the diffusion of supercoils during initial DNA unwinding. Efficient initiation therefore requires a high local concentration of topoisomerases to relax the torsional stress.
Subject(s)
Chromatin/metabolism , DNA Replication , DNA Topoisomerases, Type I/metabolism , DNA, Viral/biosynthesis , Simian virus 40 , Antigens, Viral, Tumor/metabolism , Chromatin/ultrastructure , DNA, Superhelical/metabolism , Nucleosomes/metabolism , Protein Binding , Replication OriginABSTRACT
We determined the effects of chromatin structure on template accessibility to replication factors and used three different templates as substrates for simian virus 40 (SV40) DNA replication in vitro: native and salt-treated SV40 minichromosomes and protein-free SV40 DNA. Native minichromosomes contain histone H1 and numerous nonhistone proteins in addition to the core histones, whereas salt-treated minichromosomes carry essentially only core histones. We reasoned that the less densely packed salt-treated minichromosomes should be more effective replication templates due to their more extended configuration. However, contrary to this expectation, we found that native minichromosomes replicated with significantly higher efficiency than salt-treated minichromosomes, while protein-free DNA was most active as a replication template. The higher replication efficiency of native minichromosomes was due to two activities bound to the chromatin, which were identified as DNA topoisomerases I and II. By using chromatin substrates of different general configurations, we also showed that the overall chromatin structure determines accessibility to topoisomerases I and II and thereby the efficiency of replicative chain elongation.
Subject(s)
DNA Replication , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type I/metabolism , DNA, Viral/biosynthesis , Simian virus 40/genetics , Chromatin/metabolism , HeLa Cells , Humans , Trypsin/metabolismABSTRACT
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.
Subject(s)
Chromosomes/genetics , Histones/metabolism , Simian virus 40/genetics , Virus Replication/genetics , Acetylation , Animals , Chromatin/genetics , Chromatin/metabolism , Histones/geneticsABSTRACT
We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M-phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro.
Subject(s)
Chromatin/ultrastructure , DNA Replication , DNA, Viral/ultrastructure , Histones/metabolism , Simian virus 40/genetics , Animals , Cell Cycle , Cell Line , Chlorocebus aethiops , Chromatin/metabolism , DNA, Viral/metabolism , Histones/isolation & purification , Mitosis , Phosphorylation , Resting Phase, Cell Cycle , S Phase , Simian virus 40/physiologyABSTRACT
The influence of histone H1 on DNA replication was studied using the SV40 in vitro replication system with two different templates: histone H1/DNA complexes and SV40 minichromosomes reconstituted with H1. We found that the cytosolic extracts used as a source of enzymes for in vitro replication contained high amounts of RNA which competed with template DNA for the binding of histone H1. Removal of this RNA was necessary to ensure the stability of the templates thus allowing for the first time the study of the replication of histone H1-carrying templates in vitro. In contrast to the inhibitory effect of histone H1 on the initiation of transcription, bound H1, when present at physiological ratios, does not interfere with the in vitro replication of DNA and minichromosomes. Ratios higher than one H1 molecule per nucleosome affected replication of reconstituted SV40 minichromosomes.
