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Papers In Press, published online ahead of print April 12, 2004
Molecular Biology & Human Genetics, Karmanos Cancer Institute, Detroit, MI 48201
Corresponding Author: brushg{at}karmanos.org
The cellular single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) becomes phosphorylated periodically during the normal cell cycle and also in response to DNA damage. In Saccharomyces cerevisiae, RPA phosphorylation requires the checkpoint protein Mec1, a protein kinase homologous in structure and function to human ATR. We confirm here that immunocomplexes containing a tagged version of Mec1 catalyze phosphorylation of purified RPA, likely reflecting an RPA kinase activity intrinsic to Mec1. A significant stimulation of this activity is observed upon addition of covalently closed ssDNA derived from bacteriophage M13. This stimulation is not observed with mutant RPA deficient for DNA binding, indicating that DNA-bound RPA is a preferred substrate. Stimulation is also observed upon addition of linear ssDNA homopolymers or hydrolyzed M13 ssDNA. In contrast to circular ssDNA, these DNA cofactors stimulate both wild type and mutant RPA phosphorylation. This finding suggests that linear ssDNA can also stimulate Mec1-mediated RPA phosphorylation by activating Mec1 or an associated protein. While the Mec1-interacting protein Ddc2 is required for RPA phosphorylation in vivo, it is required for neither basal nor ssDNA-stimulated RPA phosphorylation in vitro. Therefore, stimulation of Mec1-mediated RPA phosphorylation by either circular or linear ssDNA does not operate through Ddc2. Our results provide insight into the mechanisms that operate in vivo to specifically induce RPA phosphorylation upon initiation of DNA replication, repair, or recombination.
J. Biol. Chem, 10.1074/jbc.M312353200
Submitted on November 11, 2003
Revised on March 26, 2004
Accepted on April 12, 2004
DNA stimulates Mec1-mediated phosphorylation of replication protein A
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