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RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions
Gregory G Oakley1, Steve M Patrick, Jiaqin Yao
1Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Abstract:
The heterotrimeric DNA-binding protein, replication protein A (RPA), consists of 70-, 34-, and 14-kDa subunits and is involved in maintaining genomic stability by playing key roles in DNA replication, repair, and recombination. RPA participates in these processes through its interaction with other proteins and its strong affinity for single-stranded DNA (ssDNA). RPA-p34 is phosphorylated in a cell-cycle-dependent fashion primarily at Ser-29 and Ser-23, which are consensus sites for Cdc2 cyclin-dependent kinase. By systematically examining RPA-p34 phosphorylation throughout the cell cycle, we have found there are distinct phosphorylated forms of RPA-p34 in different cell-cycle stages. We have isolated and purified a unique phosphorylated form of RPA that is specifically associated with the mitotic phase of the cell cycle. The mitotic form of RPA (m-hRPA) shows no difference in ssDNA binding activity as compared with recombinant RPA (r-hRPA), yet binds less efficiently to double-stranded DNA (dsDNA). These data suggest that mitotic phosphorylation of RPA-p34 inhibits the destabilization of dsDNA by RPA complex, thereby decreasing the binding affinity for dsDNA. The m-hRPA also exhibits altered interactions with certain DNA replication and repair proteins. Using highly purified proteins, m-hRPA exhibited decreased binding to ATM, DNA pol alpha, and DNA-PK as compared to unphosphorylated recombinant RPA (r-hRPA). Dephosphorylation of m-hRPA was able to restore the interaction with each of these proteins. Interestingly, the interaction of RPA with XPA was not altered by RPA phosphorylation. These data suggest that phosphorylation of RPA-p34 plays an important role in regulating RPA functions in DNA metabolism by altering specific protein-protein interactions.
Insights
Replication protein A (RPA) phosphorylation creates a unique mitotic form (m-hRPA) that alters DNA binding and protein interactions, regulating DNA replication and repair. This mitotic RPA deactivates dsDNA binding, impacting genomic stability.
Area of Science:
- Molecular Biology
- Genomics
- Cell Biology
Background:
- Replication protein A (RPA) is a heterotrimeric DNA-binding protein crucial for genomic stability.
- RPA plays vital roles in DNA replication, repair, and recombination through protein interactions and ssDNA binding.
- RPA-p34 subunit phosphorylation occurs cell-cycle-dependently, primarily at Ser-29 and Ser-23 sites targeted by Cdc2 kinase.
Purpose of the Study:
- To systematically examine RPA-p34 phosphorylation across the cell cycle.
- To isolate and characterize a unique phosphorylated RPA form specific to mitosis.
- To investigate the functional consequences of this mitotic RPA form on DNA binding and protein interactions.
Main Methods:
- Cell cycle analysis of RPA-p34 phosphorylation.
- Isolation and purification of a unique mitotic form of RPA (m-hRPA).
- In vitro assays to compare m-hRPA and recombinant RPA (r-hRPA) for ssDNA and dsDNA binding affinities and interactions with DNA metabolism proteins (ATM, DNA pol alpha, DNA-PK, XPA).
Main Results:
- Distinct phosphorylated forms of RPA-p34 were observed at different cell cycle stages.
- A unique mitotic form of RPA (m-hRPA) was isolated, showing no change in ssDNA binding but reduced dsDNA binding compared to r-hRPA.
- m-hRPA exhibited decreased binding to ATM, DNA pol alpha, and DNA-PK, which was restored upon dephosphorylation, while XPA interaction remained unaltered.
Conclusions:
- Mitotic phosphorylation of RPA-p34 generates m-hRPA, which reduces dsDNA binding affinity, potentially preventing dsDNA destabilization during mitosis.
- Phosphorylation of RPA-p34 regulates RPA function in DNA metabolism by modulating specific protein-protein interactions.
- These findings highlight a novel regulatory mechanism for RPA activity during the cell cycle, impacting DNA replication and repair processes.