CHROMATIN STRUCTURE AND TRANSCRIPTIONAL ACTIVITY AROUND THE REPLICATION FORKS ARRESTED AT THE 3' END OF THE YEAST RIBOSOMAL-RNA GENES

Replication intermediates containing forks arrested at the replication fork barrier near the 3' end of the yeast rRNA genes were analyzed at the chromatin level by using in vivo psoralen cross-linking as a prob e for chromatin structure. These specific intermediates were purified from preparative two-dimensional agarose gels, and the extent of cross -linking in the different portions of the branched molecules was exami ned by electron microscopy and by using a psoralen gel retardation ass ay. The unreplicated section corresponding to the rRNA coding region u pstream of the arrested forks appeared mostly heavily cross-linked, ch aracteristic of transcriptionally active rRNA genes devoid of nucleoso mes, whereas the replicated daughter strands representing newly synthe sized spacer sequences showed a nucleosomal organization typical for b ulk chromatin. The failure to detect replication forks arrested at the 3' end of inactive rRNA gene copies and the fact that most DNA encodi ng rRNA (rDNA) is replicated in the same direction as transcription su ggest that replication forks seldom originate from origins of replicat ion located immediately downstream of inactive genes.