Maria Isabel Martínez-Jiménez

Primases are essential enzymes that initiate DNA replication by synthesizing de novo primers for replicative DNA polymerases, which ensure accurate genome duplication owing to their high specificity, fidelity, and processivity. PrimPols have emerged as versatile DNA-initiating enzymes that are widely distributed across diverse replication systems, from bacteriophages to humans. In this work, a novel PrimPol enzyme, PP271, encoded by bacteriophage vB_EfaS-271, was characterized. PP271 belongs to the archaeal–eukaryotic primase (AEP) superfamily and contains the conserved catalytic core, together with a C-terminal PriCT domain. Enzymatic analysis demonstrated that PP271 functions as a bona fide PrimPol, i.e. a DNA primase capable of synthetizing short DNA primers. PP271 priming activity is strongly stimulated by ADP , which serves as the preferred initiating nucleotide. Additionally, PP271 exhibits relaxed specificity for selecting the priming site, compared to other PrimPols with more restricted template preferences. TruePrime™ amplification technology combines the Thermus thermophilus PrimPol and the highly processive Phi29 DNA polymerase to achieve primer-free DNA amplification. While highly efficient, further improvements in amplification yield, fidelity and product complexity remain possible. Here, we show that DNA primers synthesized by PP271 are efficiently extended by Phi29 DNA polymerase, enabling their coordinated action. Moreover, the short primers generated by PP271 are expected to minimize the contribution of primase-derived errors to the final amplification product while allowing efficient DNA synthesis across a broader range of sequence contexts. Altogether, these findings identify PP271 as a promising alternative for improving primase-dependent DNA amplification. Key points: Bacteriophage EFAS271 encodes PP271, a DNA-dependent DNA primase-polymerase PP271 preferentially uses ADP as the initiating nucleotide during primer synthesis PP271 enables primer-free isothermal DNA amplification with Phi29 DNApol

The human PrimPol counteracts DNA replication stress by repriming DNA synthesis when fork progression is hindered by UV light or hydroxyurea treatment, or by encountering complex DNA structures, such as G-quadruplexes, R-loops, or interstrand crosslinks. The Mus musculus PrimPol (MmPrimPol) shares a high degree of amino acid similarity with its human ortholog; however, as shown here, MmPrimPol exhibits a more powerful primase activity compared to the human enzyme. Such a robust primase activity relies on an enhanced ability to bind the 5′ site nucleotide, and consequently to form initial dimers and further mature primers. Additionally, a shorter linker between the AEP core and the Zn finger domain (ZnFD) in the murine homolog likely promotes a constitutive closing of these domains into a primase-ready configuration. Consequently, a reinforced close configuration of the ZnFD would explain why MmPrimPol has a more robust primase, but a very limited DNA polymerization on an existing primer.

PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA primers ahead of blocking lesions or abnormal structures in the ssDNA template. PrimPol’s active site is formed by three AEP-conserved motifs: A, B and C. Motifs A and C of human PrimPol (HsPrimPol) harbor the catalytic residues (Asp¹¹⁴, Glu¹¹⁶, Asp²⁸⁰) acting as metal ligands, whereas motif B includes highly conserved residues (Lys¹⁶⁵, Ser¹⁶⁷ and His¹⁶⁹), which are postulated to stabilize 3′ incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys²⁹⁷, almost invariant in the whole AEP superfamily, and Lys³⁰⁰, specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His¹⁶⁹ is absolutely essential for 3′dNTP binding and, hence, for both primase and polymerase activities of HsPrimPol, whereas Ser¹⁶⁷ and Lys²⁹⁷ are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys¹⁶⁵ does not affect the overall primase function; however, it is required for damage avoidance via primer–template realignments. Finally, Lys³⁰⁰ is identified as an extra anchor residue to stabilize the 3′ incoming dNTP. Collectively, these results demonstrate that individual ligands modulate the stabilization of 3′ incoming dNTPs to optimize DNA primer synthesis efficiency during initiation and primer maturation.

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3′nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg²⁹¹ residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3′-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg²⁹¹, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3′elongation site, thus avoiding synthesis of abortive primers.

Replication forks often stall at damaged DNA. Resumption of DNA synthesis can occur by replacement of the replicative DNA polymerase with specialized, error-prone translesion DNA polymerases (TLS), that have higher tolerance for damaged substrates. Several of these polymerases (Polλ, Polη and PrimPol) are stimulated in DNA synthesis through interaction with PolDIP2, however the mechanism of this PolDIP2-dependent stimulation is still unclear. Here we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol’s enhanced processivity. PolDIP2 increases PrimPol’s primer-template and dNTP binding affinity, which concomitantly enhances PrimPol’s nucleotide incorporation efficiency. This activity is dependent on a unique arginine cluster in PolDIP2 and could be essential for PrimPol to function in vivo , since the polymerase activity of PrimPol alone is very limited. This mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, could be common to all other PolDIP2-interacting TLS polymerases, i.e. Polλ, Polη, Polζ and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.