SUMO-Targeted Ubiquitin Ligase, Rad60, and Nse2 SUMO Ligase Suppress Spontaneous Top1–Mediated DNA Damage and Genome Instability

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.

Dual Recruitment of Cdc48 (p97)-Ufd1-Npl4 Ubiquitin-selective Segregase by Small Ubiquitin-like Modifier Protein (SUMO) and Ubiquitin in SUMO-targeted Ubiquitin Ligase-mediated Genome Stability Functions

“…5C), echoing the identical rescue of slx8-29 tdp1⌬ synthetic sickness by Top1 deletion (10). The elongated phenotype of ufd1-1 tdp1⌬ cells is due to activation of the DNA damage checkpoint kinase Chk1, which blocks cell cycle progression by antagonizing CDK activation (10,39). Consistently, ufd1-1 tdp1⌬ chk1⌬ triple mutant cells were not elongated (Fig.…”

“…This phenotype was completely reversed by deleting Top1 (Fig. 5C), echoing the identical rescue of slx8-29 tdp1⌬ synthetic sickness by Top1 deletion (10). The elongated phenotype of ufd1-1 tdp1⌬ cells is due to activation of the DNA damage checkpoint kinase Chk1, which blocks cell cycle progression by antagonizing CDK activation (10,39).…”

“…Chromatin Immunoprecipitation (ChIP) Assay-Strains expressing endogenous nmt41:FLAG-Top1 were cultured at 25°C in the absence of B1 for 30 h. The cells were harvested, and native ChIP experiments without cross-linking were performed essentially as described (10). For each experiment, the percentage DNA recovery of ChIP samples relative to the DNA amount in the input was averaged over triplicate quantitative PCR measurements (Chromo4, Bio-Rad).…”

“…Elevated Levels of Spontaneous Top1cc in ufd1-1 tdp1⌬ CellsWe previously showed that elevated levels of stalled covalent Top1-DNA adducts, also known as Top1 cleavage complexes (Top1cc) cause the severe growth and genome instability phenotypes of slx8-29 tdp1⌬ cells (10). As deleting Top1 reverses the ufd1-1 tdp1⌬ phenotype, we tested whether there were increased levels of Top1cc in this background as well, which would account for activation of the G 2 DNA damage checkpoint in cells lacking Tdp1 (10).…”

“…The last finding suggests that either a polysumoylated protein(s) dominantly interferes with key cellular processes or, that the SUMO chains are themselves toxic and the critical STUbL target. Although the relevant pathways that are perturbed by STUbL inactivation remain largely undefined, STUbL maintains genome stability, at least in part, by facilitating the removal of genotoxic covalent topoisomerase I (Top1)-DNA adducts (10).…”