title: Interplay of DNA damage and cell cycle signaling at the level of human replication protein A.
abstract: Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in human cell cycle and DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-strand break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phases of the cell cycle. All three subunits of RPA contain phosphorylation sites but the exact set of HR-relevant phosphorylation sites on RPA is unknown. In this study, a high resolution capillary isoelectric focusing immunoassay, used under native conditions, revealed the isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Moreover, the phosphorylation sites of chromatin-bound and cytosolic RPA in S and G2 phases were identified by western and IEF analysis with all available phosphospecific antibodies for RPA2. Strikingly, most of the RPA heterotrimers in control G2 cells are phosphorylated with 5 isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33. DNA damaged cells in G2 had 9 isoforms with up to 14 phosphates. DNA damage isoforms contained pSer4/8, pSer12, pThr21, pSer23, and pSer33 on RPA2 and up to 8 unidentified phosphorylation sites.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: gborgstahl@unmc.edu.
authors.forename: Gloria E O
authors.initials: GE
authors.lastname: Borgstahl
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: Kerry
authors.initials: K
authors.lastname: Brader
authors.affiliation: Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68583, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Mosel
authors.affiliation: Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68583, USA.
authors.forename: Shengqin
authors.initials: S
authors.lastname: Liu
authors.affiliation: Institute for Molecular Immunology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), 81377 München, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kremmer
authors.affiliation: School of Interdisciplinary Informatics, College of Information Science and Technology, University of Nebraska at Omaha, Omaha, NE 68182, USA.
authors.forename: Kaitlin A
authors.initials: KA
authors.lastname: Goettsch
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: Carol
authors.initials: C
authors.lastname: Kolar
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Heinz-Peter
authors.initials: HP
authors.lastname: Nasheuer
authors.affiliation: Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68583, USA.
authors.forename: Greg G
authors.initials: GG
authors.lastname: Oakley
mesh_terms: D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D016195:G2 Phase; D006801:Humans; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D002843:Chromatin; D020033:Protein Isoforms; D051716:Replication Protein A
keywords: DNA damage response; DNA double-stranded breaks; DNA repair; G2 phase; Homologous recombination; Isoelectric focusing; Phosphorylation
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