DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair

Steve M. Patrick, Gregory G Oakley, Kathleen Dixon, John J. Turchi

Research output: Contribution to journalArticle

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Abstract

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the kon and k off to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase α but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)8438-8448
Number of pages11
JournalBiochemistry
Volume44
Issue number23
DOIs
StatePublished - Jun 14 2005

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Replication Protein A
DNA-Binding Proteins
DNA Replication
DNA Repair
DNA Damage
Repair
DNA
Proteins
Fluorescence Polarization
DNA-Directed DNA Polymerase
Metabolic Networks and Pathways

ASJC Scopus subject areas

  • Biochemistry

Cite this

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair. / Patrick, Steve M.; Oakley, Gregory G; Dixon, Kathleen; Turchi, John J.

In: Biochemistry, Vol. 44, No. 23, 14.06.2005, p. 8438-8448.

Research output: Contribution to journalArticle

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