Orc mutants arrest in metaphase with abnormally condensed chromosomes

Development. 2001 May;128(9):1697-707. doi: 10.1242/dev.128.9.1697.

Abstract

The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae. Our discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us to speculate that ORC may play some general role in chromosomal folding. To explore the role of ORC in chromosomal condensation, we have identified a mutant of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5) and have characterized the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43. Both Orc mutants died at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis. For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle. In addition, sister chromatid cohesion was frequently lost. PCNA and MCM4 mutants had similar phenotypes to Orc mutants. We propose that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation was observed. Thus, cells have a mitotic checkpoint that senses chromosome integrity. These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bromodeoxyuridine / metabolism
  • Cell Cycle Proteins / genetics
  • Chromosomes / ultrastructure*
  • DNA Replication*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Drosophila Proteins
  • Drosophila melanogaster / genetics*
  • Insect Proteins / metabolism
  • Metaphase*
  • Minichromosome Maintenance Complex Component 4
  • Models, Genetic
  • Mutation
  • Origin Recognition Complex
  • Proliferating Cell Nuclear Antigen / genetics
  • Saccharomyces cerevisiae Proteins*

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Drosophila Proteins
  • Insect Proteins
  • Orc2 protein, Drosophila
  • Origin Recognition Complex
  • Proliferating Cell Nuclear Antigen
  • Saccharomyces cerevisiae Proteins
  • MCM4 protein, S cerevisiae
  • Minichromosome Maintenance Complex Component 4
  • Bromodeoxyuridine