The structure of replicating simian virus 40 (SV40) minichromosomes was studied 
by DNA crosslinking with trimethyl-psoralen. The procedure was used both in 
vitro with extracted SV40 minichromosomes as well as in vivo with SV40-infected 
cells. Both procedures gave essentially the same results. Mature SV40 
minichromosomes are estimated to contain about 27 nucleosomes (error +/- 2), 
except for those molecules with a nucleosome-free gap, which are interpreted to 
contain 25 nucleosomes (error +/- 2). In replicative intermediates, nucleosomes 
are present in the unreplicated parental stem with the replication fork possibly 
penetrating into the nucleosomal DNA before the histone octamer is removed. 
Nucleosomes reassociate on the newly replicated DNA branches at distances from 
the branch point of 225 ( +/- 145) nucleotides on the leading strand and of 285( 
+/- 120) nucleotides on the lagging strand. In the presence of cycloheximide, 
daughter duplexes contained unequal numbers of nucleosomes, supporting 
dispersive and random segregation of parental nucleosomes. These were arranged 
in clusters with normal nucleosome spacing. We detected a novel type of 
interlocked dimer comprising two fully replicated molecules connected by a 
single-stranded DNA bridge. We cannot decide whether these dimers represent 
hemicatenanes or whether the two circles are joined by a Holliday-type 
structure. The joining site maps within the replication terminus. We propose 
that these dimers represent molecules engaged in strand segregation.