Mechanism and role of cooperative binding of bacteriophage fd gene 5 protein to single-stranded deoxyribonucleic acid.

The highly cooperative binding of fd gene 5 to single-stranded DNA was studied kinetically by rapid photo-cross-linking and stopped-flow UV absorption measurements. The observed change in absorbance was shown to be due to the binding by direct evidence of rapid photo-cross-linking of the bound proteins to fd DNA. The bimolecular rate constant obtained for the association was 1.6 X 10(10) M-1 s-1 (in terms of the molecular concentration of DNA), which was concluded to be diffusion controlled. The breakdown of cluster complexes on fd DNA was induced by the addition of large excess amounts of short single-stranded DNA. The breakdown took place in about 1 s. The kinetic process of redistribution of dissociated proteins was monitored by rapid photo-cross-linking and subsequent electrophoresis of the cross-linked complex. The dissociated proteins first formed isolated complexes, but later they were again converted into the cluster. The kinetic results showed that the cooperativity originated from the stabilization of the protein-DNA complex by the cluster formation, not from the accelerated association in the cluster formation. This kind of cooperative binding was shown to perform negative feedback control in the cluster formation. On the basis of the kinetic results obtained, we proposed a model for the regulatory role of the fd gene 5 protein in the synthesis of single-stranded fd DNA.