Replication initiation and elongation fork rates within a differentially expressed human multicopy locus in early S...

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• The human ribosomal RNA gene locus (rDNA) contains approximately 400 copies of a 43 kb repeat.
• Replication of this large locus spans most of the S phase, suggesting a unique regulatory mechanism.

Purpose of the Study:

• To investigate the basis for the unusual replication timing of the human rDNA locus.
• To map replication origins and measure fork progression rates within the rDNA locus.
• To correlate chromatin status and transcription with rDNA replication.

Main Methods:

• Differential intragenomic replication timing was employed to map replication origins and measure fork progression.
• A photochemical-based technique was used to assay chromatin status throughout S phase.
• Analysis focused on a 10.7 kb region encompassing the rDNA promoter and 5' external transcribed spacer.

Main Results:

• Replication initiation occurred in early S phase within a 10.7 kb region spanning the promoter and 5' external transcribed spacer.
• Bidirectional replication forks showed slow progression rates (0.32 kb/min sense, 0.23 kb/min anti-sense).
• Approximately 85% of rDNA repeats were transcriptionally active at the start of S phase, decreasing by mid-S phase.

Conclusions:

• Data suggest a link between RNA polymerase I-mediated transcription and site-specific DNA replication initiation within the rDNA locus.
• Early S phase initiation and slow fork progression may facilitate coordinated replication of the multicopy rDNA array.
• Chromatin structure and transcriptional activity dynamically regulate rDNA replication timing.