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Updated: Jul 19, 2026
08:53
Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
Published on: May 2, 2025
Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes
Sangaralingam Kumaran1, Alexander G Kozlov, Timothy M Lohman
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Biochemistry
|September 28, 2006
Summary
Saccharomyces cerevisiae replication protein A (scRPA) binds single-stranded DNA (ssDNA) in two distinct modes, influenced by salt concentration. This protein uses different numbers of oligonucleotide/oligosaccharide binding (OB)-folds depending on the binding mode.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Replication protein A (RPA) is a crucial eukaryotic protein complex essential for DNA replication, repair, and recombination.
- Understanding the DNA binding properties of RPA, particularly Saccharomyces cerevisiae RPA (scRPA), is key to elucidating its diverse cellular functions.
- Previous studies suggest RPA may interact with single-stranded DNA (ssDNA) in various configurations.
Purpose of the Study:
- To investigate the ssDNA binding modes of scRPA.
- To determine the influence of salt concentration on scRPA-ssDNA complex formation and stoichiometry.
- To characterize the thermodynamic and structural parameters of scRPA binding to ssDNA of varying lengths.
Main Methods:
- Utilized fluorescence titrations, isothermal titration calorimetry, and sedimentation equilibrium to analyze scRPA-ssDNA interactions.
- Measured parameters including occluded site size, stoichiometry, binding constants, and binding enthalpy as a function of oligodeoxynucleotide length (L) and salt concentration ([NaCl]).
Main Results:
- scRPA forms a stable heterotrimer across a wide range of salt concentrations.
- A salt-dependent transition in the occluded ssDNA site size was observed, shifting from 18-20 nucleotides at low [NaCl] to 26-28 nucleotides at high [NaCl].
- This transition correlates with a change in the stoichiometry of scRPA-(dT)L complexes and suggests the involvement of three OB-folds in the low site size mode and four OB-folds in the high site size mode.
Conclusions:
- scRPA exhibits at least two distinct ssDNA binding modes, modulated by salt concentration.
- The low salt mode utilizes three OB-folds, while the high salt mode engages four OB-folds, indicating conformational flexibility in ssDNA binding.
- While showing some similarities to E. coli SSB, scRPA's binding behavior presents unique characteristics, highlighting the diversity of ssDNA binding proteins.
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