Regulation of the translesion DNA polymerase eta by the E3 ubiquitin ligase NOPO. Heather A. Wallace¹, Julie A. Merkle², Laura A. Lee¹. 1) Cell and Developmental Biology, Vanderbilt University, Nashville, TN; 2) Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ.

   We previously 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 due to activation of a Chk2-mediated DNA checkpoint. 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 TNF Receptor Associated Factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains that closely resemble those of known E3 ubiquitin ligases. We sought to elucidate the mechanism by which NOPO/TRIP promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of non-canonical DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We have shown that Drosophila NOPO similarly interacts with Drosophila Y-family polymerase eta in cultured cells. Furthermore, we observe enhanced ubiquitination of DNA polymerase eta by TRIP and NOPO E3 ligases in cultured cells. We generated a null mutation in DNApol-eta to determine its role during Drosophila embryogenesis. Mutations in human Pol result in a variant form of xeroderma pigmentosum, a disease characterized by increased UV sensitivity and skin cancer risk. We found that both DNApol-eta and nopo-derived embryos show increased sensitivity to UV irradiation. Additionally, DNApol-eta embryos exhibit nopo-like spindle defects. We show that the decreased hatch rates and spindle defects observed in nopo-derived embryos are suppressed by overexpression of DNApol-Eta. These findings suggest that NOPO ubiquitinates DNApol-Eta and may act as a positive regulator of its activity during early embryogenesis.