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Multiple sites of replication initiation in the human beta-globin gene locus
1Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA.
Nucleic Acids Research
|February 13, 2001
Summary
Replication initiation sites in the human globin gene locus differ between normal cell growth and when protein synthesis is inhibited. Multiple initiation sites are active in logarithmically growing cells, not just one.
Area of Science:
- Genetics
- Molecular Biology
- Cell Biology
Background:
- Eukaryotic DNA replication initiation is precisely regulated, with prereplicative complexes forming in a cell cycle-dependent manner.
- While timing of replication initiation is understood, the precise location relative to prereplicative complex sites remains unclear.
- Previous studies indicated a single replication initiation site 5' to the beta-globin gene in emetine-treated cells.
Purpose of the Study:
- To investigate the pattern of DNA replication initiation sites within the human beta-globin gene locus in logarithmically growing HeLa cells.
- To compare replication initiation patterns in untreated cells versus cells with inhibited protein synthesis.
- To identify and characterize the specific genomic locations of replication origins.
Main Methods:
- Utilized two PCR-based nascent strand assays to analyze DNA replication initiation.
- Examined short nascent DNA strands across a 40 kb region of the human globin gene locus.
- Quantified nascent DNA chains to determine the abundance of initiation events at different sites.
Main Results:
- Replication initiation occurs at multiple sites within the beta-globin gene locus in logarithmically growing HeLa cells, unlike the single site observed in emetine-treated cells.
- Identified several active replication origins, including one near the previously defined initiation region (IR).
- Discovered a major initiation site approximately 20 kb upstream of the IR, showing 4-fold greater nascent strand abundance, and this site is adjacent to an early S phase replicating fragment.
Conclusions:
- The location of DNA replication initiation within the human globin gene locus is flexible and context-dependent, varying with cellular conditions.
- Logarithmically growing cells utilize multiple replication origins within this domain, suggesting a more complex initiation landscape than previously thought under drug treatment.
- The identified major upstream initiation site represents a significant origin of replication in actively dividing cells.