no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila

Development. 2009 Feb;136(3):449-59. doi: 10.1242/dev.027599.

Abstract

In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named ;no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Checkpoint Kinase 2
  • DNA Damage
  • Drosophila / embryology
  • Drosophila / enzymology*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Drosophila Proteins / physiology*
  • Embryo, Nonmammalian / physiology
  • Female
  • HeLa Cells
  • Humans
  • Mitosis
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Protein Serine-Threonine Kinases / genetics
  • Spindle Apparatus / genetics
  • Spindle Apparatus / ultrastructure
  • Two-Hybrid System Techniques
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / physiology*

Substances

  • Drosophila Proteins
  • Ubiquitin-Conjugating Enzymes
  • ben protein, Drosophila
  • NOPO protein, Drosophila
  • Ubiquitin-Protein Ligases
  • Checkpoint Kinase 2
  • Protein Serine-Threonine Kinases
  • lok protein, Drosophila