Single-Molecule Movies of the Interplay between DNA Polymerase and Single Strand DNA Binding (SSB) Protein

Abstract

Reliable DNA replication is completed by the orchestrated activation of a multi-protein machinery, the replisome, in a highly coordinated and dynamic fashion. The bacteriophage T7 gene 2.5 protein (gp2.5) works as a single strand DNA(ssDNA) binding protein and has been demonstrated to play a key role in the maintenance of ssDNA stability as well as the suppression of hairpin formation during each cycle of Okazaki fragment synthesis. After gp2.5 binding to ssDNA, gp2.5 might act as a road block for DNA polymerase to proceed. Bulk biochemical assays, however, demonstrate that the presence of gp2.5 increases the processivity and rate of template extension, while a mutant gp2.5 without c-terminus decreases the activity. Here we present movies of the single-molecule dynamics of T7 DNA polymerase and gp2.5 using optical trapping combined with confocal fluorescence microscopy. First, we investigated at single molecule level the effect of presence of wide type and mutant gp2.5 on DNA polymerase activity under various conditions. Then, we observed in real-time the labelled gp2.5 binding and unbinding dynamics interacting with DNA polymerase. It demonstrates that gp2.5 binds with ssDNA at low force (<30pN) but that its binding to ssDNA decreases significantly at higher force (>30pN). Beside, fluorescence data shows labelled DNA polymerase appearing at multiple places on the DNA which indicates the DNA polymerase might bind to DNA at multiple sites. These results combined with further visualization of the two labeled proteins will help us better understand the interplay between DNA polymerase and gp2.5.