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DNA stimulates Mec1-mediated phosphorylation of replication protein A
Amy J Bartrand1, Dagmawi Iyasu, George S Brush
1Program in Molecular Biology and Human Genetics, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
Abstract:
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 the addition of covalently closed ssDNA derived from the 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 the 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. Although 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, activation of Mec1-mediated RPA phosphorylation by either circular or linear ssDNA does not operate through Ddc2. Our results provide insight into the mechanisms that function in vivo to specifically induce RPA phosphorylation upon initiation of DNA replication, repair, or recombination.
Insights
Replication protein A (RPA) phosphorylation is regulated by the Mec1 kinase. Both circular and linear single-stranded DNA (ssDNA) stimulate this process, but through different mechanisms, revealing new insights into DNA replication and repair.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Replication protein A (RPA) is a crucial cellular protein that binds single-stranded DNA (ssDNA).
- RPA undergoes phosphorylation during the cell cycle and in response to DNA damage.
- Mec1, a protein kinase in Saccharomyces cerevisiae, is essential for RPA phosphorylation and is homologous to human ATR.
Purpose of the Study:
- To investigate the intrinsic kinase activity of Mec1 towards RPA.
- To determine the role of single-stranded DNA (ssDNA) in stimulating Mec1-mediated RPA phosphorylation.
- To elucidate the involvement of the Mec1-interacting protein Ddc2 in this process.
Main Methods:
- In vitro kinase assays using purified RPA and Mec1 immunocomplexes.
- Assessment of RPA phosphorylation in the presence of various forms of ssDNA (circular, linear, hydrolyzed).
- Analysis of RPA phosphorylation using wild-type and DNA-binding deficient RPA mutants.
Main Results:
- Mec1 directly phosphorylates RPA in vitro, indicating an intrinsic RPA kinase activity.
- Covalently closed circular ssDNA significantly stimulates Mec1-mediated RPA phosphorylation, particularly with DNA-binding proficient RPA.
- Linear ssDNA and hydrolyzed circular ssDNA also stimulate RPA phosphorylation, suggesting activation of Mec1 or associated proteins.
- Ddc2, while essential in vivo, is not required for basal or ssDNA-stimulated RPA phosphorylation in vitro.
Conclusions:
- Mec1 possesses intrinsic RPA kinase activity, modulated by ssDNA.
- Both circular and linear ssDNA can activate Mec1-mediated RPA phosphorylation, employing distinct mechanisms.
- The Ddc2 protein is not directly involved in the ssDNA-mediated activation of Mec1's RPA kinase activity in vitro.
- These findings offer mechanistic insights into the regulation of RPA phosphorylation during DNA replication, repair, and recombination.
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