title: Disruption of SUMO-targeted ubiquitin ligases Slx5-Slx8/RNF4 alters RecQ-like helicase Sgs1/BLM localization in yeast and human cells.
abstract: RecQ-like helicases are a highly conserved protein family that functions during DNA repair and, when mutated in humans, is associated with cancer and/or premature aging syndromes. The budding yeast RecQ-like helicase Sgs1 has important functions in double-strand break (DSB) repair of exogenously induced breaks, as well as those that arise endogenously, for example during DNA replication. To further investigate Sgs1's regulation, we analyzed the subcellular localization of a fluorescent fusion of Sgs1 upon DNA damage. Consistent with a role in DSB repair, Sgs1 recruitment into nuclear foci in asynchronous cultures increases after ionizing radiation (IR) and after exposure to the alkylating agent methyl methanesulfonate (MMS). Yet, despite the importance of Sgs1 in replicative damage repair and in contrast to its elevated protein levels during S-phase, we find that the number of Sgs1 foci decreases upon nucleotide pool depletion by hydroxyurea (HU) treatment and that this negative regulation depends on the intra S-phase checkpoint kinase Mec1. Importantly, we identify the SUMO-targeted ubiquitin ligase (STUbL) complex Slx5-Slx8 as a negative regulator of Sgs1 foci, both spontaneously and upon replicative damage. Slx5-Slx8 regulation of Sgs1 foci is likely conserved in eukaryotes, since expression of the mammalian Slx5-Slx8 functional homologue, RNF4, restores Sgs1 focus number in slx8 cells and furthermore, knockdown of RNF4 leads to more BLM foci in U-2 OS cells. Our results point to a model where RecQ-like helicase subcellular localization is regulated by STUbLs in response to DNA damage, presumably to prevent illegitimate recombination events.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Böhm
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
authors.forename: Michael Joseph
authors.initials: MJ
authors.lastname: Mihalevic
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
authors.forename: Morgan Alexandra
authors.initials: MA
authors.lastname: Casal
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA. Electronic address: karab@pitt.edu.
authors.forename: Kara Anne
authors.initials: KA
authors.lastname: Bernstein
mesh_terms: D000293:Adolescent; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D059767:Recombinational DNA Repair; D059807:S Phase Cell Cycle Checkpoints; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C114070:RNF4 protein, human; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; C515574:Slx8 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; C097151:Bloom syndrome protein; C090472:SGS1 protein, S cerevisiae; D053484:RecQ Helicases; C515573:Slx5 protein, S cerevisiae
keywords: BLM; RNF4; STUbL; Sgs1; Slx5–Slx8