DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage

Jonathan E. Nuss, Steve M. Patrick, Greg G. Oakley, Gerald M. Alter, Jacob G. Robison, Kathleen Dixon, John Turchi

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.

Original languageEnglish (US)
Pages (from-to)8428-8437
Number of pages10
JournalBiochemistry
Volume44
Issue number23
DOIs
StatePublished - Jun 14 2005
Externally publishedYes

Fingerprint

Replication Protein A
Phosphorylation
DNA Damage
DNA
Gels
Phospho-Specific Antibodies
Aphidicolin
Hydroxyurea
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Electrophoresis, Gel, Two-Dimensional
DNA-Binding Proteins
Electrophoresis
DNA Replication
HeLa Cells
DNA Repair
Genetic Recombination
Polyacrylamide Gel Electrophoresis
Digestion
Cell Cycle
Repair

ASJC Scopus subject areas

  • Biochemistry

Cite this

DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage. / Nuss, Jonathan E.; Patrick, Steve M.; Oakley, Greg G.; Alter, Gerald M.; Robison, Jacob G.; Dixon, Kathleen; Turchi, John.

In: Biochemistry, Vol. 44, No. 23, 14.06.2005, p. 8428-8437.

Research output: Contribution to journalArticle

Nuss, Jonathan E. ; Patrick, Steve M. ; Oakley, Greg G. ; Alter, Gerald M. ; Robison, Jacob G. ; Dixon, Kathleen ; Turchi, John. / DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage. In: Biochemistry. 2005 ; Vol. 44, No. 23. pp. 8428-8437.
@article{75bd93bd939b4909b6b2d1a4c2471b4c,
title = "DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage",
abstract = "Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.",
author = "Nuss, {Jonathan E.} and Patrick, {Steve M.} and Oakley, {Greg G.} and Alter, {Gerald M.} and Robison, {Jacob G.} and Kathleen Dixon and John Turchi",
year = "2005",
month = "6",
day = "14",
doi = "10.1021/bi0480584",
language = "English (US)",
volume = "44",
pages = "8428--8437",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "23",

}

TY - JOUR

T1 - DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage

AU - Nuss, Jonathan E.

AU - Patrick, Steve M.

AU - Oakley, Greg G.

AU - Alter, Gerald M.

AU - Robison, Jacob G.

AU - Dixon, Kathleen

AU - Turchi, John

PY - 2005/6/14

Y1 - 2005/6/14

N2 - Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.

AB - Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.

UR - http://www.scopus.com/inward/record.url?scp=20444375871&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=20444375871&partnerID=8YFLogxK

U2 - 10.1021/bi0480584

DO - 10.1021/bi0480584

M3 - Article

C2 - 15938632

AN - SCOPUS:20444375871

VL - 44

SP - 8428

EP - 8437

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 23

ER -