Role of Tyr-22 in the binding of Pf3 ssDNA binding protein to nucleic acids.

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.