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Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.

Stephanie M Cohen1, Seigo Hatada, Bruna P Brylawski

  • 1Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA. stepmi@med.unc.edu

Genomics
|October 23, 2004
PubMed
Summary
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Researchers identified a functional DNA replication origin within the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene. This origin’s activity was restored in cells lacking it by introducing specific human DNA sequences.

Area of Science:

  • Molecular Biology
  • Genetics
  • Genomics

Background:

  • DNA replication origins are crucial for genome duplication.
  • The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus has been investigated for its role in replication.
  • Understanding replication origin function is key to comprehending genome stability.

Purpose of the Study:

  • To map a functional origin of DNA replication.
  • To investigate the role of the HPRT gene promoter and exon 1 in replication origin activity.
  • To identify cis-acting elements involved in mammalian origin selection and activation.

Main Methods:

  • Functional mapping of replication origins using quantitative PCR on nascent DNA.
  • Analysis of DNA deletions in mouse embryonic stem (ES) cells.

Related Experiment Videos

  • Homologous recombination to restore gene activity and replication origin function.
  • Sequence analysis of human and mouse DNA for cis-acting elements.

    • Main Results:

    • A functional replication origin was localized to the HPRT gene's promoter and exon 1 in mouse and human genomes.
    • Replication origin activity was lost in ES cells with a deletion at the 5' end of the HPRT locus.
    • Restoration of HPRT activity via homologous recombination reconstituted origin activity.
    • Initiation of DNA replication was shown to coincide with the insertion site of human DNA containing HPRT exon 1 and a truncated promoter.

    Conclusions:

    • The HPRT gene promoter and exon 1 region contain a functional DNA replication origin.
    • Specific DNA sequences, including human HPRT exon 1 and promoter elements, can confer replication origin activity.
    • Further analysis is needed to identify the precise cis-acting elements responsible for origin function and selection.