The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication

George S. Brush, Carl W. Anderson, Thomas J. Kelly

Research output: Contribution to journalArticle

Abstract

The 32-kDa subunit of replication protein A (RPA) is phosphorylated during the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of this modification, we purified a HeLa cell protein kinase that phosphorylates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated protein kinase (DNA-PK), a previously described high molecular weight protein kinase that is capable of phosphorylating a number of nuclear DNA binding proteins. Phosphorylation of RPA by DNA-PK is stimulated by natural single-stranded DNAs but not by homopolymers lacking secondary structure. Studies with the simian virus 40 model system indicate that DNA-PK is required for DNA-replication-dependent RPA phosphorylation. Depletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phosphorylation of RPA by DNA-PK is activated by formation of replication intermediates containing single- and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replication with the cell cycle.

Original languageEnglish (US)
Pages (from-to)12520-12524
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number26
DOIs
StatePublished - Dec 20 1994

ASJC Scopus subject areas

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