RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions

Gregory G. Oakley, Steve M. Patrick, Jiaqin Yao, Michael P. Carty, John J. Turchi, Kathleen Dixon

Research output: Contribution to journalArticle

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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 α, 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.

Original languageEnglish (US)
Pages (from-to)3255-3264
Number of pages10
JournalBiochemistry
Volume42
Issue number11
DOIs
StatePublished - Mar 25 2003

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Replication Protein A
Phosphorylation
Mitosis
Carrier Proteins
Proteins
Cell Cycle
Cells
Recombinant proteins
Recombinant Proteins
Repair
Recombinational DNA Repair
Cyclin-Dependent Kinases
Genomic Instability
Single-Stranded DNA
DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Oakley, G. G., Patrick, S. M., Yao, J., Carty, M. P., Turchi, J. J., & Dixon, K. (2003). RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions. Biochemistry, 42(11), 3255-3264. https://doi.org/10.1021/bi026377u

RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions. / Oakley, Gregory G.; Patrick, Steve M.; Yao, Jiaqin; Carty, Michael P.; Turchi, John J.; Dixon, Kathleen.

In: Biochemistry, Vol. 42, No. 11, 25.03.2003, p. 3255-3264.

Research output: Contribution to journalArticle

Oakley, GG, Patrick, SM, Yao, J, Carty, MP, Turchi, JJ & Dixon, K 2003, 'RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions', Biochemistry, vol. 42, no. 11, pp. 3255-3264. https://doi.org/10.1021/bi026377u
Oakley, Gregory G. ; Patrick, Steve M. ; Yao, Jiaqin ; Carty, Michael P. ; Turchi, John J. ; Dixon, Kathleen. / RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions. In: Biochemistry. 2003 ; Vol. 42, No. 11. pp. 3255-3264.
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