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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.

Biochemistry
|March 19, 2003
PubMed

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.

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