no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila.
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.
Merkle
Julie A
JA
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-4200 MRBIII, 465 21st Avenue South, Nashville, TN 37232, USA.
Rickmyre
Jamie L
JL
Garg
Aprajita
A
Loggins
Erin B
EB
Jodoin
Jeanne N
JN
Lee
Ethan
E
Wu
Louisa P
LP
Lee
Laura A
LA
D000595:Amino Acid Sequence; D000818:Animals; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004330:Drosophila / Q000196:embryology / Q000201:enzymology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004625:Embryo, Nonmammalian / Q000502:physiology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D008941:Spindle Apparatus / Q000235:genetics / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000502:physiology*
D029721:Drosophila Proteins; D044763:Ubiquitin-Conjugating Enzymes; C471544:ben protein, Drosophila; C540453:NOPO protein, Drosophila; D044767:Ubiquitin-Protein Ligases; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C112435:lok protein, Drosophila