Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A.
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 (15)N relaxation experiments were performed on an isolated ssDNA-binding domain (RPA70A) from the human SSB replication protein A. The backbone (13)C, (15)N, and (1)H 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-C(5) 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 (R(1) and R(2)) and (1)H-(15)N 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.
Bhattacharya
Shibani
S
Department of Biochemistry, and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725, USA.
Botuyan
Maria-Victoria
MV
Hsu
Fred
F
Shan
Xi
X
Arunkumar
A I
AI
Arrowsmith
Cheryl H
CH
Edwards
Aled M
AM
Chazin
Walter J
WJ
D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding / Q000502:physiology; D051716:Replication Protein A
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051716:Replication Protein A