A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes

Abstract

Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time. The gp32 protein probe displayed high sensitivity and specificity to a variety of ssDNA-dependent processes of T4 phage. Several applications of the probe are illustrated here: the solution dynamics of ssDNA-binding protein, protein-protein and protein-DNA interactions involving gp32 protein and its mode of interaction, ssDNA translocation and protein displacement activities of helicases, primer extension activity of DNA polymerase holoenzyme and nucleoprotein filament formation during DNA recombination. The assay has identified new protein-protein interactions of gp32 during T4 replication and recombination. The fluorescent probe described here can thus be used as a universal probe for monitoring in real time various ssDNA-dependent processes, which is based on a well-characterized and easy-to-express bacteriophage T4 gene 32 protein, gp32.