Unraveling the Interplay between Single-Stranded DNA-Binding Protein, DNA Polymerase and Single-Stranded DNA

Abstract

Bacteriophage T7 gene 2.5 protein (gp2.5), a single-stranded DNA binding (SSB) protein, contains two main structural elements which confer it essential functions. First, an oligosaccharide/oligonucleotide binding fold which can interact with ssDNA and thus provide a protective role while removing secondary structure impediments. Second, an acidic C-terminal tail capable to interact with other gp2.5 units and with other replisome proteins, such as the T7 DNA primase-helicase (gp4) and polymerase (gp5), allowing for organizational roles within the replisome. The impact of SSBs on the replisome dynamics is hard to study using conventional biochemistry tools. Here, we present data obtained from a unique combination of optical tweezers and confocal fluorescence microscopy, which offers real-time high spatial and temporal resolution to study protein-DNA interactions. Our results show that the SSB gp2.5 binds efficiently to ssDNA, forming highly static protein-DNA complexes. We also observed a decrease in the ssDNA end-to-end length indicating that the protein binds by bending or wrapping ssDNA, resulting in a highly tension-sensitive binding mode. Next, we studied to what degree the presence of wild type (wt) gp2.5 or a mutant lacking the terminal phenylalanine (ΔF), the interaction motif with DNA polymerase, affects replication activity. We show that wt gp2.5 alters the rate, duration and probability of occurrence of replicative and proofreading polymerase events in contrast to the ΔF mutant or the absence of the protein. Thus, our findings indicate that the presence of gp2.5 strongly stimulates the function of DNA polymerase within the replisome.