REPLICATION INITIATION AND ELONGATION FORK RATES WITHIN A DIFFERENTIALLY EXPRESSED HUMAN MULTICOPY LOCUS IN EARLY S-PHASE

Replication of the 400 copies of the 43 kb human ribosomal RNA (rDNA) locus spans most of the S phase, To examine the basis for the unusual pattern of rDNA replication, a sensitive strategy was developed to map origins of DNA replication and measure apparent rates of fork progres sion within a chromosomal locus. This technique, termed differential i ntragenomic replication timing, revealed that initiation within the ac tively transcribed rDNA occurred in early S within a 10.7 kb region sp anning the promoter and 5' external transcribed spacer, Forks emanatin g from this early bidirectional origin progressed at an apparent slow rate with the sense and anti-sense forks moving at 0.32 and 0.23 kb/mi n. Using a photochemical-based technique, the chromatin status of the rDNA repeats was assayed throughout the S phase. Approximately 85% of the rDNA repeats were in a transcriptionally active chromatin structur e at the start of S phase. A progressive decrease in the transcription state of the rDNA loci was observed, reaching a minimum between 3 and 6 h in mid S phase. Altogether, the data suggest a link between RNA p olymerase I mediated transcription and site-specific initiation of DNA replication within the rDNA multicopy locus.