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.

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

“…S4 B). Moreover, although overexpression of TRA IP WT stimulated polη polyubiquitylation as previously reported (Wallace et al, 2014), this depended neither on its RING nor PIP domains (Fig. S4 C), and depletion of TRA IP did not reduce polη ubiquitylation levels (Fig.…”

“…TRA IP has recently been linked to other genome integrity maintenance processes, including TLS through ubiquitylation of the TLS polymerase polη and in the spindle assembly checkpoint (Chapard et al, 2014;Wallace et al, 2014). However, we found that these involvements were unlikely to explain the requirement of TRA IP in genome maintenance after replication stress.…”

“…The TLS polymerase polη has been recently suggested as one such target, showing enhanced recruitment to nuclear foci in cells overexpressing TRA IP (Wallace et al, 2014). However, our data collectively suggest that polη is unlikely to be a major effector of TRA IP in the responses to replication stress described in this study.…”

“…However, we found that these involvements were unlikely to explain the requirement of TRA IP in genome maintenance after replication stress. Although overexpression of TRA IP has been shown to enhance polη recruitment to DNA damage sites (Wallace et al, 2014), knockdown of TRA IP had no impact on the formation of DNA damage-induced polη foci (Fig. S4 B).…”

“…TRA IP has previously been implicated in NF-κB signaling, cell proliferation, and the spindle checkpoint (Chapard et al, 2012(Chapard et al, , 2014. Recently, TRA IP was also suggested to regulate the translesion DNA synthesis polymerase polη when overexpressed (Wallace et al, 2014), but whether this represents a primary function of TRA IP in the DDR is not known. We therefore set out to explore the functional significance of its association with damaged DNA.…”