View the Current Issue

Article

  • The EMBO Journal (2006) 25, 5150 - 5158
  • doi:10.1038/sj.emboj.7601391

Published online: 19 October 2006

An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes

Jeffrey W Semple1, Lance F Da-Silva1, Eric J Jervis2, Jennifer Ah-Kee3, Hyder Al-Attar1, Lutz Kummer1, John J Heikkila1, Philippe Pasero3 and Bernard P Duncker1

  1. Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
  2. Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada
  3. Institute of Human Genetics, Centre National de la Recherche Scientifique, Montpellier Cedex, France

Correspondence to:

Bernard P Duncker, Department of Biology, University of Waterloo, 200 University Ave., Waterloo, Ontario, Canada N2L 3G1. Tel.: +1 519 888 4567 x 33957; Fax: +1 519 746 0614; E-mail: bduncker@sciborg.uwaterloo.ca

Received 5 May 2006; Accepted 19 September 2006

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.

  • Keywords:

    • DNA replication,
    • minichromosome maintenance protein,
    • origin recognition complex,
    • pre-replicative complex