Regulation of Drosophila early embryogenesis and genome maintenance by the E3 ubiquitin ligase no poles
Merkle, Julie Ann
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 showed 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 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 completion of DNA replication. NOPO is the Drosophila homolog of mammalian TRAF-interacting protein (TRIP). NOPO and TRIP contain highly similar RING domains that closely resemble that of known E3 ubiquitin ligases. We showed that Bendless (BEN), an E2 ubiquitin conjugating enzyme, interacts with NOPO; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitylation complex consisting of BEN/UEV1A and NOPO is required for preservation of genomic integrity during early embryogenesis. We sought to elucidate the mechanism by which NOPO/TRIP promotes genomic stability by performing a yeast two-hybrid screen to identify NOPO/TRIP interactors. We identified a family of non-canonical DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. Furthermore, we have shown that NOPO interacts with Drosophila Y polymerases and we observe an enhanced ubiquitylation of the Y-family polymerases by TRIP and NOPO E3 ligases. To determine if Y polymerases have a role in Drosophila early embryogenesis, we generated a null mutation in DNApol-eta and observe decreased hatch rates and nopo-like spindle defects in DNApol-eta-derived embryos. Mutation of the human homolog, POLH, results in a variant form of Xeroderma Pigmentosum, a disease characterized by UV sensitivity and skin cancer. We hypothesize that DNApol-eta has a unique role during Drosophila early embryogenesis to promote cell-cycle progression and that NOPO regulates its activity.