Initiation and termination of DNA replication in human rRNA genes.
We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enrichment for replicating molecules was achieved by isolating S-phase enriched populations of cells by centrifugal elutriation, purification of DNA associated with the nuclear matrix, and a chromatographic procedure that enriches for molecules containing single-stranded regions, a characteristic of replication forks. Two-dimensional agarose gel electrophoresis techniques were used to demonstrate that replication appears to initiate at multiple sites throughout most of the 31-kb nontranscribed spacer (NTS) of human ribosomal DNA but not within the 13-kb transcription unit or adjacent regulatory elements. Although initiation events were detected throughout the majority of the NTS, some regions may initiate more frequently than others. Termination of replication, the 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 site-specific termination of replication is the result of pausing of replication forks near the sites of transcription termination. The naturally occurring multicopy rRNA gene family offers a unique system to study mammalian DNA replication without the use of chemical synchronization agents.
Little
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RD
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Platt
T H
TH
Schildkraut
C L
CL
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004261:DNA Replication; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D012335:RNA, Ribosomal / Q000235:genetics*; D015183:Restriction Mapping
D012335:RNA, Ribosomal