Minichromosome replication in vitro: inhibition of re-replication by replicatively assembled nucleosomes.
Single-stranded circular DNA, containing the SV40 origin sequence, was used as a template for complementary DNA strand synthesis in cytosolic extracts from HeLa cells. In the presence of the replication-dependent chromatin assembly factor CAF-1, defined numbers of nucleosomes were assembled during complementary DNA strand synthesis. These minichromosomes were then induced to semiconservatively replicate by the addition of the SV40 initiator protein T antigen (re-replication). The results indicate that re-replication of minichromosomes appears to be inhibited by two independent mechanisms. One acts at the initiation of minichromosome re-replication, and the other affects replicative chain elongation. To directly demonstrate the inhibitory effect of replicatively assembled nucleosomes, two types of minichromosomes were prepared: (i) post-replicative minichromosomes were assembled in a reaction coupled to replication as above; (ii) pre-replicative minichromosomes were assembled independently of replication on double-stranded DNA. Both types of minichromosomes were used as templates for DNA replication under identical conditions. Replicative fork movement was found to be impeded only on post-replicative minichromosome templates. In contrast, pre-replicative minichromosomes allowed one unconstrained replication cycle, but re-replication was inhibited due to a block in fork movement. Thus, replicatively assembled chromatin may have a profound influence on the re-replication of DNA.
Krude
T
T
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Knippers
R
R
D002498:Centrifugation; D002843:Chromatin / Q000502:physiology; D002877:Chromosomes, Human; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics; D004279:DNA, Viral; D006367:HeLa Cells; D006801:Humans; D009707:Nucleosomes; D013539:Simian virus 40 / Q000235:genetics
D002843:Chromatin; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009707:Nucleosomes