Mechanistic Studies of DNA-Protein Interactions in Bacteriophage T4 DNA Replication Complexes at Single-Base Resolution

Abstract

Combining biophysical measurements on the function and control of T4 bacteriophage replication complexes with detailed structural information can throw light onto the mechanism of action of these ‘macromolecular machines’. In this study we use the low energy circular dichroism and fluorescent properties of site-specifically introduced fluorescent base analogues to monitor the binding and interactions of gene 32 protein (gp32), the ssDNA binding protein of the T4 DNA replication complex, and gene 59 protein (gp59), the helicase loader protein involved in loading the T4 primosome complex onto replication forks. We show that gp32 binds preferentially to the 3'-end of a short ssDNA oligomer and that only two or three nucleotide bases at the 3' end are directly perturbed by the binding interaction, although the binding site size in the polynucleotide binding mode is 7 nts. Loss of exciton coupling in the CD spectrum and an increase in fluorescence intensity shows that gp32 binding causes extension of the sugar-phosphate backbone of the oligonucleotide. Mechanisms involved in gp32 binding to its ssDNA targets in both the isolated and the cooperative binding modes will be described. In addition our CD and fluorescence experiments with base analogue probes show the gp59 binding to a forked DNA construct perturbs the bases at the junction and suggests that once gp41 is loaded the perturbation extends deep into the double helix. Mechanisms whereby these proteins work in tandem in setting up various functional interactions with the other components of the DNA replication complex will be discussed.