The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction

Sara K Binz; Ye Lao; David F Lowry; Marc S Wold

doi:10.1074/jbc.M305388200

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The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction

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The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction

Sara K Binz, Ye Lao, David F Lowry and Marc S Wold

The Journal of biological chemistry, Vol.278(37), pp.35584-35591

09/12/2003

DOI: https://doi.org/10.1074/jbc.M305388200

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https://hdl.handle.net/2376/114040

PMID: 12819197

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Abstract

Recombinant Proteins - metabolism

Sequence Deletion

Phosphorylation

Molecular Weight

Mutagenesis, Site-Directed

Protein Structure, Secondary

DNA, Single-Stranded - metabolism

Models, Molecular

DNA Replication

DNA-Binding Proteins - chemistry

Protein Subunits - metabolism

DNA-Binding Proteins - metabolism

Peptide Fragments - chemistry

Animals

Protein Conformation

DNA Damage

Replication Protein A

Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.

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Title: The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction
Creators: Sara K Binz - Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242-1109, USA
Ye Lao
David F Lowry
Marc S Wold
Publication Details: The Journal of biological chemistry, Vol.278(37), pp.35584-35591
Academic Unit: Engineering and Applied Sciences (TRIC), School of
Publisher: United States
Grant note: GM4471 / NIGMS NIH HHS
Identifiers: 99900547776801842
Language: English
Resource Type: Journal article