Mapping of amino acid residues in the p34 subunit of human single- stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro

Hongwu Niu, Hediye Erdjument-Bromage, Zhen Qiang Pan, Suk-Hee Lee, Paul Tempst, Jerard Hurwitz

Research output: Contribution to journalArticle

73 Citations (Scopus)

Abstract

Human single-stranded DNA-binding protein (HSSB, also called RPA), is a heterotrimeric complex that consists of three subunits, p70, p34, and p11. HSSB is essential for the in vitro replication of SV40 DNA and nucleotide excision repair. It also has important functions in other DNA transactions, including DNA recombination, transcription, and double-stranded DNA break repair. The p34 subunit of HSSB is phosphorylated in a cell cycle-dependent manner. Both Cdc2 kinase and the DNA-dependent protein kinase (DNA-PK) phosphorylate HSSB-p34 in vitro. In this study, we show that efficient phosphorylation of HSSB-p34 by DNA-PK requires Ku as well as DNA. The DNA-PK phosphorylation sites in HSSB-p34 have been mapped at Thr-21 and Ser-33. Kinetic studies demonstrated that a phosphate residue is first incorporated at Thr-21 followed by the incorporation of a second phosphate residue at Ser- 33. We also identified Ser-29 as the major Cdc2 kinase phosphorylation site in the p34 subunit.

Original languageEnglish (US)
Pages (from-to)12634-12641
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number19
DOIs
StatePublished - May 9 1997
Externally publishedYes

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DNA-Activated Protein Kinase
DNA-Binding Proteins
Phosphotransferases
Phosphorylation
Amino Acids
DNA Repair
DNA
Phosphates
Double-Stranded DNA Breaks
DNA Replication
Repair
Genetic Recombination
Cell Cycle
Transcription
Nucleotides
Cells
In Vitro Techniques
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mapping of amino acid residues in the p34 subunit of human single- stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro. / Niu, Hongwu; Erdjument-Bromage, Hediye; Pan, Zhen Qiang; Lee, Suk-Hee; Tempst, Paul; Hurwitz, Jerard.

In: Journal of Biological Chemistry, Vol. 272, No. 19, 09.05.1997, p. 12634-12641.

Research output: Contribution to journalArticle

Niu, Hongwu ; Erdjument-Bromage, Hediye ; Pan, Zhen Qiang ; Lee, Suk-Hee ; Tempst, Paul ; Hurwitz, Jerard. / Mapping of amino acid residues in the p34 subunit of human single- stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 19. pp. 12634-12641.
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