title: TRIP/NOPO E3 ubiquitin ligase promotes ubiquitylation of DNA polymerase η.
abstract: We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, U-4225 Medical Research Building III, 465 21st Avenue South, Nashville, TN 37232-8240, USA.
authors.forename: Heather A
authors.initials: HA
authors.lastname: Wallace
authors.forename: Julie A
authors.initials: JA
authors.lastname: Merkle
authors.forename: Michael C
authors.initials: MC
authors.lastname: Yu
authors.forename: Taloa G
authors.initials: TG
authors.lastname: Berg
authors.forename: Ethan
authors.initials: E
authors.lastname: Lee
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bosco
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lee
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D030801:Animals, Genetically Modified; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D042822:Genomic Instability; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D015398:Signal Transduction; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D029721:Drosophila Proteins; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D044767:Ubiquitin-Protein Ligases; C540453:NOPO protein, Drosophila; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
keywords: Cell cycle; DNA damage; Drosophila; E3 ubiquitin ligase; Embryogenesis; Xeroderma pigmentosum