Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A

Authors

  • Shibani Bhattacharya,

    1. Departments of Biochemistry and Physics, and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725, USA
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  • Maria-Victoria Botuyan,

    1. Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada
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  • Fred Hsu,

    1. Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada
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  • Xi Shan,

    1. Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada
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  • A.I. Arunkumar,

    1. Departments of Biochemistry and Physics, and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725, USA
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  • Cheryl H. Arrowsmith,

    1. Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada
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  • Aled M. Edwards,

    1. Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada
    2. Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada
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  • Walter J. Chazin

    Corresponding author
    1. Departments of Biochemistry and Physics, and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725, USA
    • Departments of Biochemistry and Physics, and Center for Structural Biology, 5142 BIOSCI/MRBIII, Vanderbilt University, Nashville, TN 37232-8725, USA; fax: (615) 936-2211.
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Abstract

Single-stranded-DNA-binding proteins (SSBs) are required for numerous genetic processes ranging from DNA synthesis to the repair of DNA damage, each of which requires binding with high affinity to ssDNA of variable base composition. To gain insight into the mechanism of sequence-nonspecific binding of ssDNA, NMR chemical shift and 15N relaxation experiments were performed on an isolated ssDNA-binding domain (RPA70A) from the human SSB replication protein A. The backbone 13C, 15N, and 1H resonances of RPA70A were assigned for the free protein and the d-CTTCA complex. The binding-induced changes in backbone chemical shifts were used to map out the ssDNA-binding site. Comparison to results obtained for the complex with d-C5 showed that the basic mode of binding is independent of the ssDNA sequence, but that there are differences in the binding surfaces. Amide nitrogen relaxation rates (R1 and R2) and 1H–15N NOE values were measured for RPA70A in the absence and presence of d-CTTCA. Analysis of the data using the Model-Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA-binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, we propose that the mechanism of sequence-nonspecific binding of ssDNA involves dynamic remodeling of the binding surface.

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