G Nigel Godson, PhD

We are studying the structure and mechanism of action of Escherichia coli primase and human primase. This critically important enzyme is required to initiate DNA replication in all prokaryote and eukaryote cells. It does this by synthesizing a small RNA chain (primer RNA) on the DNA template which provides a free 3 OH group for DNA polymerase to use to copy the complementary DNA strand. Both prokaryote and eukaryote primases share many features in common and our experimental results obtained with Escherichia coli primase can often be translated to human primase.

All primases lack mechanisms to keep them attached to the DNA template and consequently only synthesize short stretches of primer RNA. We are developing evidence for a model of action of Escherichia coli primase that we think is applicable to all primases. We have shown that it syntheses pRNA in short steps. Once it has synthesized the first step, it disengages from the template and a second primase molecule attaches and continues the primer chain extension. This cycle can be repeated indefinitely in the absence of other proteins that limit its reinitiation.

To support this model, we are studying the structure of primase and its interaction with the template DNA. We have established that Escherichia coli primase consists of three physical domains: 1) an N-terminal 110-amino acid domain, containing a zinc finger which probably interacts with the DNA template; 2) an internal 322-amino acid domain containing the catalytic center where the actual polymerization steps take place and 3) a C-terminal 151-amino acid domain that is involved in protein-protein contacts. We are identifying the major functional features of primase, such as the template binding site and the catalytic center and are analyzing how they function in the different stages of pRNA synthesis. Similar studies of human primase are underway.