Mechanism of T7 Primase Selecting Active Priming Sites Among Genome

In bacteriophage T7, the primase synthesizes primer at a specific site, 5'-(G/T)₂GTC-3'. However, the pentanucleotide alone cannot define the activity of the primase. In this study, we demonstrated that the 10-nt sequence flanking the 3' end of pentanucleotide sites made considerable contributions to the interactions between T7 primase and single-strand DNA (ssDNA). Approximately 26 template sequences with multiple features were screened out from the T7 genome, which exhibited strong binding affinity to T7 primase and high priming activity, thus supporting genome replication. Notably, a dinucleotide in the 3' flank of the pentanucleotide site was found to be instrumental in T7 primase binding to ssDNA, which might be recognized by the zinc-binding domain of T7 primase. As a result, a multiple-site recognition model for T7 primase to select priming sites was proposed. These results shed light on how T7 primase selects priming sites, a process that may be shared by its bacterial counterparts. Furthermore, our study provides novel methodologies for investigating the interactions between prokaryotic primases and their ssDNA templates, thereby laying the groundwork for the development of novel inhibitors.