Crystal Structure And Biochemical Studies Of The Deep-sea Vent Phage DNA Polymerase

A novel DNA polymerase from the deep-sea vent phage Nr S-1,was characterized as a primase-polymerase?referred to as Prim-Pol?,which works as a self-priming DNA polymerase to synthesize de novo long DNA strands.Functional research on the Nr S-1Prim-Pol illustrated that the N-terminal 300 residues?referred to as N300?have de novo synthesis activity similar to that of the full-length enzyme.Here,the crystal structure of N300 was solved at a resolution of 1.80?,and the three kinds of crystal structures of N300-dNTPs-Mg²⁺complex also were determined.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?.These N300 complex structures shared the identical steric architecture and hydrogen-bond interactions in the catalytic center.Both the prim/pol domain of N300 and other Prim-Pols 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.The results of biochemical studies also indicated that R145 possibly plays an indispensable role in the primer extension.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.Just like other Prim-Pols,N300 was able to initiate DNA synthesis,proficiently discriminating against ribonucleotides?NTPs?,exclusively using deoxynucleotides?dNTPs?.However,the structural basis for this discrimination is not well understood.Mutagenesis and structural simulation showed that the backbone carboxyl group of Y146,as a potential sugar selector,was involved in steric clashing with the incoming2?-OH group of NTPs.However,the mechanism of substrate discrimination probably was different from that of other Prim-Pols,according to the structural analyses and sequence comparison.The structure and biochemical studies of N300 would give overall insights into the functional specificity,catalytic mechanism and evolutionary relationship of Prim-Pols.