Abstract
Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)–mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl–DNA phosphodiesterase 1 (Tdp1). This is notable, as cells lacking Tdp1 are largely aphenotypic in the vegetative cell cycle due to the existence of alternative pathways for the removal of covalent Top1–DNA adducts (Top1cc). We further identify Rad60, a SUMO mimetic and STUbL-interacting protein, and the SUMO E3 ligase Nse2 as critical Top1cc repair factors in cells lacking Tdp1. Detection of Top1ccs using chromatin immunoprecipitation and quantitative PCR shows that they are elevated in cells lacking Tdp1 and STUbL, Rad60, or Nse2 SUMO ligase activity. These unrepaired Top1ccs are shown to cause DNA damage, hyper-recombination, and checkpoint-mediated cell cycle arrest. We further determine that Tdp1 and the nucleotide excision repair endonuclease Rad16-Swi10 initiate the major Top1cc repair pathways of fission yeast. Tdp1-based repair is the predominant activity outside S phase, likely acting on transcription-coupled Top1cc. Epistasis analyses suggest that STUbL, Rad60, and Nse2 facilitate the Rad16-Swi10 pathway, parallel to Tdp1. Collectively, these results reveal a unified role for STUbL, Rad60, and Nse2 in protecting genome stability against spontaneous Top1-mediated DNA damage.
Highlights
Efficient DNA repair suppresses spontaneous genetic alterations that otherwise lead to cell death or transformation
Much is known about specific DNA repair mechanisms, an understanding of how these processes are critically orchestrated by post-translational modifiers such as SUMO and ubiquitin is in its infancy
We identified an intriguing family of E3 ubiquitin ligases called SUMO-targeted ubiquitin ligase (STUbL) that act at the interface between the SUMO and ubiquitin pathways, and through undefined proteins and pathways maintain genome stability
Summary
Efficient DNA repair suppresses spontaneous genetic alterations that otherwise lead to cell death or transformation. Posttranslational modifications (PTMs) can enhance the efficiency of individual repair processes and proteins and/or channel repair through appropriate pathways [1,2]) Among these PTMs, the small proteins ubiquitin and SUMO have gained increasing recognition as key guardians of chromosomal integrity [1,2,3]. Related enzymatic cascades covalently attach either SUMO or ubiquitin to lysine residues within target proteins to modulate their stability, activity and localization [3]. Each cascade employs dedicated E1 activating enzymes, E2 conjugating enzymes and E3 ligases that contribute to substrate selection and transfer of the modifier from the E2 to the target protein [3]. In contrast to the numerous ubiquitin E3 ligases, there are apparently two major SUMO E3 ligases in fission yeast called Pli and Nse2 [4,5]
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