Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase.

Fengtao Huang,Xueling Lu,Chunxiao Yu,Longfei Wang,Bin Zhu,Piotr Sliz

doi:10.3389/fmicb.2021.766612

Abstract

PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA de novo.

Highlights

PrimPols are a class of primases possessing both the primase and DNA polymerase activities
The structure of N-terminal 300 amino acid residues (N300) consists of two domains: a primase-like (Prim/Pol) domain located at the N-termini and a helix bundle domain (HBD) at the C-termini (Figures 1A,B)
It is traditionally accepted that DNA replication requires both DNA polymerase and primase in all domains of life

Summary

Introduction

PrimPols are a class of primases possessing both the primase and DNA polymerase activities. The enzymes are first identified from archaeal plasmid pRN1 (Lipps et al, 2003) and has subsequently been found in various organisms including bacteria, bacteriophages, and humans (Halgasova et al, 2012; Sanchez-Berrondo et al, 2012; García-Gómez et al, 2013; Wan et al, 2013; Picher et al, 2016; Zhu et al, 2017; Gupta et al, 2019) Based on their structures and sequences, PrimPols are grouped into the archaeo-eukaryotic primase (AEP) superfamily (Iyer et al, 2005; Guilliam et al, 2015; Kazlauskas et al, 2018). As the HBD is conserved in the AEP superfamily, it is likely to play a general role in all AEP superfamily proteins (Boudet et al, 2019)

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Conclusion