INITIATION AND TERMINATION OF DNA-REPLICATION IN HUMAN RIBOSOMAL-RNA GENES

We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enri chment for replicating molecules was achieved by isolating S-phase enr iched populations of cells by centrifugal elutriation, purification of DNA associated with the nuclear matrix, and a chromatographic procedu re that enriches for molecules containing single-stranded regions, a c haracteristic of replication forks. Two-dimensional agarose gel electr ophoresis techniques were used to demonstrate that replication appears to initiate at multiple sites throughout most of the 31-kb nontranscr ibed spacer (NTS) of human ribosomal DNA but not within the 13-kb tran scription unit or adjacent regulatory elements. Although initiation ev ents were detected throughout the majority of the NTS, some regions ma y initiate more frequently than others. Termination of replication, th e convergence of opposing replication forks, was found throughout the ribosomal DNA repeat units, and, in some repeats, specifically at the junction of the 3' end of the transcription unit and the NTS. This sit e-specific termination of replication is the result of pausing of repl ication forks near the sites of transcription termination. The natural ly occurring multicopy rRNA gene family offers a unique system to stud y mammalian DNA replication without the use of chemical synchronizatio n agents.