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The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40
G S Brush1, C W Anderson, T J Kelly
1Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
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.
Insights
The DNA-activated protein kinase (DNA-PK) phosphorylates replication protein A (RPA) during DNA replication. This modification, crucial for cell cycle coordination, is activated by replication intermediates.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Replication
Background:
- Replication protein A (RPA) is phosphorylated during S phase and viral DNA replication.
- The functional significance of RPA phosphorylation remains to be fully elucidated.
Purpose of the Study:
- To identify the kinase responsible for RPA phosphorylation.
- To investigate the role of RPA phosphorylation in DNA replication and cell cycle coordination.
Main Methods:
- Purification of a HeLa cell protein kinase.
- Identification of the kinase as DNA-activated protein kinase (DNA-PK).
- In vitro phosphorylation assays using RPA and single-stranded DNA.
Main Results:
- DNA-PK phosphorylates the 32-kDa subunit of RPA.
- Phosphorylation is stimulated by natural single-stranded DNA, not homopolymers.
- DNA-PK is required for DNA replication-dependent RPA phosphorylation in the SV40 system.
- Kinase depletion does not affect DNA replication extent in vitro.
Conclusions:
- RPA phosphorylation by DNA-PK is activated by replication intermediates with single- and double-stranded regions.
- This phosphorylation event may signal cell cycle coordination with DNA replication.