Current and emerging assays for studying the primer synthesis activities of DNA primases

Primases play a crucial role in the initiation of DNA synthesis during replication by de novo synthesis of short RNA or DNA “primers.” In recent years, evidence has accumulated which expands the essential roles of primases to include, not only the initiation of replication but also other critical roles in DNA metabolism, including damage tolerance and repair. Despite the broadening roles for these enzymes, the methods used to identify and characterize primase activities are limited. Historically, biochemical analysis of primases has been based on the synthesis of radioactively labeled primers and their detection on denaturing polyacrylamide gels. In the last two decades, a number of alternative primase assays have been developed in an effort to supersede radioactive methods. However, the radioactive gel-based assay, which has not significantly changed since its conception in the late 1970s, remains the most widely used and favored method. In this chapter, we discuss the background to, and the advantages and disadvantages of, the current techniques used to characterize primase activity in vitro. Finally, we describe an alternative, gel-based, fluorescent primase assay, which we have successfully used in the characterization of a recently identified primase-polymerase, PrimPol.