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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Dec 20;91(26):12520–12524. doi: 10.1073/pnas.91.26.12520

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

G S Brush 1, C W Anderson 1, T J Kelly 1
PMCID: PMC45470  PMID: 7809070

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.

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Selected References

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