Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

Abstract

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.

Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks

“…The template strand contains a 15-nt poly(deoxythymidine) [p(dT) 15 ] sequence that can form an association complex with up to two cooperatively bound gp32 protein monomers at one time. Recently, Lee et al (15) showed that smFRET signals observed from this same p/t DNA construct in the presence of gp32 undergo intermittent protein-induced fluctuations, which reflect changes in the end to end distance of the ssDNA template caused by binding and dissociation of gp32 proteins.…”

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments

“…As indicated above, we illustrate our approach using the macromolecular system studied by Lee et al , 8 in which a ssDNA template interacts with the T4 bacteriophage gp32 binding protein (see Fig. 1A).…”

“…Experimentally, this requires making measurements at a higher time resolution than that used in the Lee et al study. 8 As the time resolution of a single-molecule fluorescence measurement approaches a few milliseconds, the signal will necessarily become too noisy to extract the state of the system through direct visualization of single-molecule trajectory data (e.g., by HMM analysis). Rather, we show below how equivalent information may be obtained through the application of the generalized concepts of TCFs.…”

“…In a recent study from our laboratory, 8 smFRET experiments were employed to analyze the cooperative binding of the single-stranded (ss) DNA binding protein of the T4 bacteriophage DNA replication complex (gp32) to single-stranded segments of primer-template (p/t) DNA constructs of varying lengths and polarities. These constructs can serve as models of DNA replication forks.…”

“…As background, we note that gp32 protein molecules bind cooperatively and preferentially to ssDNA, with a binding site size of 7 nucleotide residues (nts, or DNA lattice positions) per gp32 molecule. 28 We have shown 8 that p/t DNA substrates with a ssDNA ‘tail’ region of 15 nts in length, which can cooperatively bind up to two gp32 proteins, can undergo stochastic fluctuations between 0-, 1- and 2-bound states (see Fig. 1A).…”