Role of Tyr-22 in the Binding of Pf3 ssDNA Binding Protein to Nucleic Acids

Abstract

A Tyr-22-->Phe-22 (Y22F) mutant of the single-strand DNA binding protein (ssDBP) of the filamentous phage Pf3 was obtained by site-directed mutagenesis. An alignment of protein sequences indicates that Tyr-22 of the Pf3 ssDBP corresponds to Tyr-26 of the fd g5p, a tyrosine within the DNA-binding loop. The mutant Y22F Pf3 protein had a CD spectrum very similar to that of native, wild-type Pf3 ssDBP and could bind to both DNA and RNA polymers. In CD titrations of poly[r(A)], poly[r(C)], and Pf3 ssDNA with the Y22F mutant, the saturation endpoints remained the same as for titrations performed with wild-type Pf3 ssDBP, indicating that the mutant protein retained the same n = 2 mode of binding as the wild-type protein. However, a second stoichiometric mode of binding at a ratio of one protein monomer to about four nucleotides (n = 4) was observed for titrations of these nucleic acids with the Y22F mutant protein. Both proteins showed only an n = 2 mode of binding to poly[d(A)], poly[d(C)], and poly[d(T)] and only an n = 3 mode of binding to poly[r(U)]. Distinctly different CD spectral changes of the nucleic acid were observed in titrations of poly[d(A)] with the Y22F mutant and the wild-type protein. Therefore, the mutant and wild-type ssDBP interact differently with some nucleic acids, depending on the base and sugar composition, providing evidence that Tyr-22 is indeed in the DNA-binding loop and may be important in the sequence discrimination of the binding of the Pf3 ssDBP.

Refined Structure, DNA Binding Studies, and Dynamics of the Bacteriophage Pf3 Encoded Single-Stranded DNA Binding Protein,

“…CD measurements on complexes formed by wild-type Pf3 ssDBP and various polynucleotides indicated a stoichiometry of n ) 2 (Powell & Gray, 1993), but the authors also found evidence for n ) 4 binding in gel electrophoresis experiments. A recent CD study on a mutant (Tyr22 f Phe) confirms the presence of this n ) 4 mode (Powell & Gray, 1995). The model shown in Figure 10 is clearly incompatible with an n ) 2 stoichiometry: the average distance between phosphorus atoms in the calculated complexes is 5.9 ( 0.9 Å.…”

Circular Dichroism Spectroscopy of Three Tyrosine-to-Phenylalanine Substitutions of fd Gene 5 Protein

“…Additionally, the ssDNA binding proteins of bacteriophages Pf 1 and Pf3 probably form DNA binding wings similar to that of fd g5p. with Tyr-30 of the Pf 1 protein and Tyr-22 of the Pf3 protein being equivalent to Tyr-26 of fd g5p (Plyte & Kneale, 1993: Powell & Gray. 1995.…”