Crystal structure and biochemical studies of the bifunctional DNA primase-polymerase from phage NrS-1.

A novel DNA polymerase found in the deep-sea vent phage NrS-1, was confirmed to have both DNA polymerase and primase activities. In this polymerase, the N-terminal residues 1-300 (referred to as N300) are the core region required for polymerizing DNA and catalyzing de novo DNA synthesis. Here, the crystal structure of N300 was solved at a resolution of 1.80 Å. The overall structure consists of a prim/pol domain and a helix bundle domain, which are separated by a 14-residue-long flexible tether (residues 177-190). Both the prim/pol domain of N300 and other primase-polymerases (prim-pol) encompass an analogous fold with conserved catalytic residues. Mutagenesis and enzymatic activity assays show that the acidic active-site residue E139 is required for both polymerase and primase activities. Functional assays confirm the essentiality of the helix bundle domain for primase activity. Furthermore, we identified a mutant (N300-Y261A) of the helix bundle domain, which probably plays an indispensable role in the primer initiation and recognition of template DNA.