Interplay of DNA damage and cell cycle signaling at the level of human replication protein A

Gloria E Borgstahl, Kerry Brader, Adam Mosel, Shengqin Liu, Elisabeth Kremmer, Kaitlin A. Goettsch, Carol Kolar, Heinz Peter Nasheuer, Gregory G Oakley

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)12-23
Number of pages12
JournalDNA Repair
Volume21
DOIs
StatePublished - Jan 1 2014

Fingerprint

Replication Protein A
DNA Damage
Phosphorylation
Cell Cycle
Cells
Protein Isoforms
DNA
Homologous Recombination
G2 Phase
S Phase
Phosphates
Phospho-Specific Antibodies
Double-Stranded DNA Breaks
DNA-Binding Proteins
Isoelectric Focusing
Post Translational Protein Processing
human RPA1 protein
Immunoassay
Metabolism
Chromatin

Keywords

  • DNA damage response
  • DNA double-stranded breaks
  • DNA repair
  • G2 phase
  • Homologous recombination
  • Isoelectric focusing
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Interplay of DNA damage and cell cycle signaling at the level of human replication protein A. / Borgstahl, Gloria E; Brader, Kerry; Mosel, Adam; Liu, Shengqin; Kremmer, Elisabeth; Goettsch, Kaitlin A.; Kolar, Carol; Nasheuer, Heinz Peter; Oakley, Gregory G.

In: DNA Repair, Vol. 21, 01.01.2014, p. 12-23.

Research output: Contribution to journalArticle

Borgstahl, Gloria E ; Brader, Kerry ; Mosel, Adam ; Liu, Shengqin ; Kremmer, Elisabeth ; Goettsch, Kaitlin A. ; Kolar, Carol ; Nasheuer, Heinz Peter ; Oakley, Gregory G. / Interplay of DNA damage and cell cycle signaling at the level of human replication protein A. In: DNA Repair. 2014 ; Vol. 21. pp. 12-23.
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