Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA.
The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.
Mou
T C
TC
Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688, USA.
Gray
C W
CW
Terwilliger
T C
TC
Gray
D M
DM
D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D017912:Inovirus / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D011061:Poly A / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012492:Salts / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism*; D017186:Titrimetry; D014443:Tyrosine / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D010755:Organophosphates; D012492:Salts; D013873:Thionucleotides; D014764:Viral Proteins; C030570:oligodeoxyadenylic acid; C068293:gene 5 protein, Enterobacteria phage T4; D011061:Poly A; C015465:poly(dA); D014443:Tyrosine; D012965:Sodium Chloride; D010649:Phenylalanine