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Related Experiment Videos

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.

I Chesnokov1, M Gossen, D Remus

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720 USA.

Genes & Development
|May 27, 1999
PubMed
Summary

Researchers identified unique genes for the origin recognition complex (ORC) in Drosophila. This suggests DNA replication origins aren't solely regulated by diverse ORC protein families, as the reconstituted complex restored replication in depleted extracts.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • DNA replication initiation in eukaryotes depends on the origin recognition complex (ORC) binding to DNA.
  • Understanding ORC's role is crucial for comprehending genome duplication and regulation.

Purpose of the Study:

  • To clone and characterize the genes encoding the subunits of the Drosophila ORC.
  • To investigate the role of ORC subunit diversity in regulating DNA replication origin usage.

Main Methods:

  • Cloning of genes encoding Drosophila ORC subunits.
  • Chromosomal mapping of identified genes.
  • Reconstitution of the six-subunit ORC using recombinant proteins.
  • Assay of reconstituted ORC activity in depleted egg extracts.

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Main Results:

  • All genes encoding Drosophila ORC subunits were uniquely identified.
  • Each gene mapped to distinct chromosomal locations.
  • The reconstituted six-subunit ORC restored DNA replication in ORC-depleted Drosophila and Xenopus egg extracts.

Conclusions:

  • Drosophila ORC is composed of unique subunits encoded by distinct genes.
  • The regulation of DNA replication origin usage is not solely dependent on a large family of ORC proteins.
  • The reconstituted ORC complex is functionally active and capable of supporting DNA replication.