Circular duplex DNA containing the SV40 replication origin was assembled into 
chromosomes in vitro with core histones and nucleosome assembly factor from HeLa 
cells. Their ability to serve as a template for replication was examined by 
incubating them with SV40 T antigen and HeLa cell extract. Nucleosome assembly 
of the template prevented DNA replication. Replication of chromosomes was 
severely inhibited at more than two-thirds of physiological nucleosome density. 
When the DNA was preincubated with T antigen and then assembled into 
chromosomes, however, inhibition of DNA replication was greatly reduced. These 
results suggest that nucleosome assembly of the template inhibits initiation of 
SV40 DNA replication and that the inhibition can be overcome by formation of an 
initiation complex before nucleosome assembly.