title: Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells.
abstract: SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational repair. We have also shown that deficiency of POLD3 leads to an increase in RPA-bound ssDNA when cells are under replication stress, suggesting that POLD3 plays a role in the cellular response to DNA replication stress. Considering that DNA replication stress is a source of genome instability, and that excessive replication stress is a hallmark of pre-neoplastic and tumor cells, our characterization of SUMO2 targets during a perturbed S-phase should provide a valuable resource for future functional studies in the fields of DNA metabolism and cancer biology.
authors.affiliation: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark.
authors.forename: Sara
authors.initials: S
authors.lastname: Bursomanno
authors.affiliation: Department of Proteomics, The Novo Nordisk Foundation Centre for Protein Research, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark; Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Beli
authors.affiliation: Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Asif M
authors.initials: AM
authors.lastname: Khan
authors.affiliation: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark.
authors.forename: Sheroy
authors.initials: S
authors.lastname: Minocherhomji
authors.affiliation: Department of Proteomics, The Novo Nordisk Foundation Centre for Protein Research, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark.
authors.forename: Sebastian A
authors.initials: SA
authors.lastname: Wagner
authors.affiliation: Department of Disease Biology, The Novo Nordisk Foundation Centre for Protein Research, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark.
authors.forename: Simon
authors.initials: S
authors.lastname: Bekker-Jensen
authors.affiliation: Department of Disease Biology, The Novo Nordisk Foundation Centre for Protein Research, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Mailand
authors.affiliation: Department of Proteomics, The Novo Nordisk Foundation Centre for Protein Research, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark.
authors.forename: Chunaram
authors.initials: C
authors.lastname: Choudhary
authors.affiliation: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark; Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Ian D
authors.initials: ID
authors.lastname: Hickson
authors.affiliation: Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark; Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. Electronic address: ying@sund.ku.dk.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
mesh_terms: D002478:Cells, Cultured; D043283:Chromosome Fragile Sites; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D040901:Proteomics; D051716:Replication Protein A / Q000378:metabolism; D016196:S Phase; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D013312:Stress, Physiological / Q000235:genetics; D058207:Sumoylation
chemical_list: D004277:DNA, Single-Stranded; D019384:Nucleic Acid Synthesis Inhibitors; D051716:Replication Protein A; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D004247:DNA; C435720:POLD3 protein, human; D004258:DNA Polymerase III
keywords: Common fragile sites; DNA replication stress; Mass spectrometry; POLD3; SUMOylation