A four-in-one replicase integrating key enzymatic activities for DNA replication

Abstract

DNA replication is a fundamental process in all living organisms. As the most diverse and abundant biological entities on Earth, bacteriophages may utilize unconventional methods for genome replication. In this study, we identified a novel DNA replicase, GP55, from lactococcal phage 1706. GP55 comprises a helicase domain, a distinctive archaeo-eukaryotic primase domain, and a family B DNA polymerase domain, collectively exhibiting helicase, primase, and DNA polymerase activities, along with intrinsic 3'-5' exonuclease activity. Notably, the helicase activity of GP55 is UTP/dTTP-dependent rather than ATP-dependent and facilitates strand displacement during DNA synthesis. GP55 exhibits a unique primase activity, recognizing specific but less stringent DNA sequences and preferring GTP for the initiation of RNA primer synthesis. Additionally, a newly identified α-helix domain, composed of two pairs of parallel α-helices, was found to be essential for its primase activity. The multiple activities enable GP55 to efficiently synthesize DNA de novo in the presence of dNTPs and NTPs. This study reveals a concise strategy employed by bacteriophages for genome replication using multifunctional replicases.

Graphical Abstract

Figure 1.

Sequence analysis showing that GP55 is a large multi-domain replicase. (A) The domain architecture of GP55. (B) Predicted structural model of GP55 generated using AlphaFold2. (C) Multiple sequence alignment of the GP55 helicase domain with those of other characterized helicase domain-containing proteins, based on [23]. (D) Multiple sequence alignment of the GP55 PriS-like domain with other PrimPol proteins, based on [7]. (E) Multiple sequence alignment of the GP55 PolB domain with other characterized PolB DNA polymerases, based on [5]. (F) Schematic illustration of the constructed GP55 mutants. (G) SDS–PAGE analysis of the purified GP55 and its mutants.

Figure 2.

The DNA polymerase activity, 3′–5′ exonuclease activity, and helicase activity of GP55. (A) Extension of ³²P-labeled 16/36-mer primer/template duplex substrate by GP55 and its mutants. (B) Primer extension by various amounts of GP55. (C) Primer extension by GP55 and GP55exo- in the presence of different concentrations of dNTPs. Samples in panels (A)–(C) were examined by 10% denaturing PAGE containing 7 M urea. (D) DNA unwinding assays catalyzed by GP55 in the presence of different kinds of NTPs or dNTPs. (E) Comparative analysis of DNA unwinding activity between GP55 and its mutants [ΔC(689–1317) and K189A-exo-]. Reaction mixtures were terminated by adding 6× Gel Loading Dye containing 0.48% SDS (New England Biolabs) in panels (D) and (E). (F) Strand displacement activity of GP55 examined using two different DNA substrates.

Figure 3.

RNA primer synthesis and DNA de novo synthesis by GP55. (A) Oligonucleotide synthesis on ssM13 DNA by GP55 in the presence of various concentrations of NTPs or dNTPs. Prominent bands were indicated by stars. (B) Time course of oligonucleotide synthesis by GP55 in the presence of 0.5 mM NTPs or 0.5 mM dNTPs. Prominent bands were indicated by stars. (C) Comparison of the RNA synthesis by GP55 and its mutants (GP55exo-, GP55-D509A, and GP55-D949A). The reactions were performed at 37°C for 15 min in the presence of 0.5 mM NTPs. (D) DNA de novo synthesis on ssM13 DNA by GP55 in the presence of various combinations of NTPs and dNTPs. C: circular M13 dsDNA; L: linear M13 dsDNA. The purchased circular ssM13 DNA contains a small amount of linear ssM13 DNA, which was replicated by GP55 to generate linear M13 dsDNA. (E) GP55-replicated dsDNA products confirmed by cleavage of EcoRI (New England Biolabs) or NdeI (New England Biolabs). The M13 DNA contains one EcoRI cleavage site (5′-GAATTC-3′) and three NdeI cleavage sites (5′-CATATG-3′). M13 dsDNA was synthesized by GP55 in the presence of 10 nM ssM13 DNA, 0.5 mM NTPs, and 0.5 mM dNTPs. The ssM13 DNA and the synthesized dsDNA products were digested with 10 U of EcoRI and NdeI, respectively.

Figure 4.

Dissection of the primer synthesis by GP55. (A) Identification of the initiation site of primer synthesis using [γ-³²P]ATP labeling. RNA primers were synthesized by GP55 on various templates in the presence of 100 μM GTP, UTP, CTP, and ∼0.4 μM [γ-³²P]ATP. The released inorganic pyrophosphate (PPi) indicated RNA primer synthesized by GP55. (B) Confirmation of the initiation site using [α-³²P]ATP labeling. RNA primers were synthesized by GP55 on the templates T51–T55 in the presence of 100 μM GTP, UTP, and CTP, and 0.19 μM [α-³²P]ATP. Marker M1, M2, and M3 were synthesized by N300 domain of NrSPol as described previously [7]. (C) Analysis of primer synthesis on the template T56. The oligonucleotide primers were synthesized by 50 nM GP55 or ΔC(689–1317) in the presence of different combinations of nucleotides (ATP, dATP, GTP, and dGTP) at 100 μM. Lanes 1–7 contain samples labeled with [α-³²P]GTP, while lanes 8–14 contain samples labeled with [α-³²P]ATP. Marker M4 was synthesized by N300 domain of NrSPol as described previously [7]. The * indicates phosphorothioate modification at the 3′ terminus of T56. (D) RNA primer synthesis coupled with DNA synthesis by GP55. RNA primer or DNA products were synthesized on template T56 by GP55, ΔC(689–1317), or the combined action of GP55-D509A and ΔC(689–1317), in the presence or absence of 100 μM ATP or 500 μM dATP.

Figure 5.

Characterization of the aHD of GP55. (A) Comparison of the RNA primer synthesis activity of GP55 and its mutants on template T56. (B) Structural superposition of GP55 primase domain with the truncated mutant (N300) of NrSPol, a PrimPol protein (PDB: 6A9W). (C) Structural comparison of the GP55 aHD, the N300 HBD, and ORF904 HBD (PDB: 3M1M). (D) Sequence alignment of the GP55 aHD and its homologous sequences. Arrowheads indicated the conserved residues selected for functional analysis. (E) SDS–PAGE analysis of the purified GP55 and its aHD-inactivated mutants. (F) Comparison of the primer synthesis activity of GP55 and its aHD-inactivated mutants. (G) Structural mapping of the key residues identified in aHD.

Figure 6.

Characterization of DNA replication by GP55 and comparison with commercial DNA polymerases. (A) De novo DNA replication on ssM13 DNA template by GP55 in the presence of dNTPs and a single type of NTP. (B) Time course analysis of DNA replication on the ssM13 DNA by GP55 in the presence of dNTPs and either all four NTPs or GTP alone. (C) Alkaline (denaturing) agarose gel analysis of the DNA products synthesized by GP55 at different time points. (D) Comparison of the de novo DNA synthesis of GP55, its mutants, and commercial DNA polymerases (T4 and Phi29) on ssM13 DNA template. Reactions contained 10 nM ssM13 DNA, 500 μM dNTPs and 500 μM NTPs, and 50 nM GP55 or its mutant, 3 U T4 (New England Biolabs) or 10 U Phi29 (New England Biolabs). (E) DNA extension activities on singly primed ssM13 DNA by GP55, its mutants, and commercial DNA polymerases (T4 and Phi29). Reactions contained 10 nM ssM13 DNA, 200 nM primer M2 (5′-CCCAGTCACGACG*T*T-3′), 500 μM dNTPs and 500 μM NTPs, 50 nM GP55 or its mutant, 3 U T4, or 10 U Phi29. (F) Examination of RNA primers synthesized by GP55 mutants [GP55-D949A and ΔC(689–1317)] and their extension by GP55, its mutants, and commercial DNA polymerases (T4 and Phi29).