Binding and reversible denaturation of double-stranded DNA by Ff gene 5 protein.
The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+).
Mou
Tung-Chung
TC
Department of Molecular and Cell Biology, Mail Stop FO31, University of Texas at Dallas, P.O. Box 830688, Richardson, TX 75083-0688, USA.
Shen
Michelle C
MC
Terwilliger
Thomas C
TC
Gray
Donald M
DM
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D009691:Nucleic Acid Denaturation; D014443:Tyrosine / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; D014443:Tyrosine; D004247:DNA