Binding of the Single-Stranded DNA Binding Protein (gp32) of T4 Bacteriophage Induces Position-Specific Local Conformational Changes in DNA Lattices that can be Monitored by Fluorescent Probes

The single-stranded (ss) DNA binding protein (gp32) of the bacteriophage T4 DNA replication system plays a major role in integrating the functions of the sub-assemblies of the replication complex by interacting with different replication proteins. Substitution of fluorescent base analogues at defined positions in nucleic acid constructs can be used to study local conformational changes of the nucleic acid bases at specific sites, and Cy3/Cy5 dye-pairs inserted into the sugar-phosphate backbones can track the backbone motions of the DNA lattice during gp32 binding. By monitoring the fluorescence, circular dichroism, absorbance and FRET signals of these base and backbone probes, equilibrium and dynamic measurements can provide information about the molecular details of replication mechanisms. In the present study we use 6-methyl isoxanthopterin (6-MI), a fluorescent base analogue of guanine, to monitor local conformational changes of DNA lattices on gp32 binding. We find that aromatic amino acid residues of gp32 stacks with the bases of ssDNA lattices, depending on the specific positions of the bases within the binding domain of the gp32 molecule, and that Molecular Dynamics simulations are consistent with the experimental results. These results may shed light on how the hydrophobic pockets found in many protein-nucleic acid interaction complexes may stabilize the intercalation or stacking of aromatic amino acid residues with nucleic acid bases. Using internally labeled cyanine dyes, we have also investigated the double-stranded DNA melting properties of gp32 protein at a replication fork junction, and our results suggest that gp32 can enhance breathing at a fork junction if the ssDNA arm at the fork is of sufficient length to permit formation of cooperatively-bound gp32 clusters at the junction.