Repetitive Single-Molecule FRET Fluctuations upon T4 Gene 32 Protein Binding to Single-Stranded DNA

Abstract

Bacteriophage T4-coded gene 32 protein (gp32) is an essential component of the T4 DNA replication, recombination, and repair systems. Gp32 is a single-stranded DNA binding protein that binds cooperatively to single-stranded (ss) DNA templates exposed by the function of the T4 primosome helicase, and configures these templates for efficient use by the replisome. To this end gp32 binding helps melt out transiently formed ssDNA secondary structures, protects exposed ssDNA sequences from nucleases, and helps to integrate the functions of the other components of the replication complex. It has been well studied by bulk biochemical methods for about 40 years. However, many aspects of the detailed mechanisms -- and especially the dynamics -- of the interactions of gp32 with both double-stranded (ds) and single-stranded DNA are still not well studied or well understood.We have used single-molecule Forster Resonance Energy Transfer (sm-FRET) methods to monitor the cooperative and non-cooperative binding of gp32 to the ssDNA sequences of model DNA replication forks, and have shown that the observed binding dynamics depend significantly on gp32 concentration, salt concentration and ssDNA segment length. Upon addition of 1 µM concentrations of gp32 into a sample chamber containing tethered single molecule replication fork constructs, we observed the appearance of ‘repetitive FRET fluctuations’ of the individual DNA molecules (>70% of the molecules per imaging area) on a 100 ms timescale. We noted also that these repetitive FRET fluctuations were substantially less prominent under the conditions of tight and fully cooperative gp32 protein binding. We have used ensemble and single-molecule fluorescence approaches to probe the dynamics of these gp32-ssDNA interactions in detail, and will discuss possible molecular interpretations of these smFRET fluctuations in terms of potential reaction intermediates and association-dissociation pathways in real time.