Search Results (4)

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. Full article

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. Full article

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC₁-LigC₂-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain. Full article

The complex molecular machines responsible for genome replication encounter many obstacles during their progression along DNA. Tolerance of these obstructions is critical for efficient and timely genome duplication. In recent years, primase-polymerase (PrimPol) has emerged as a new player involved in maintaining eukaryotic replication fork progression. This versatile replicative enzyme, a member of the archaeo-eukaryotic primase (AEP) superfamily, has the capacity to perform a range of template-dependent and independent synthesis activities. Here, we discuss the emerging roles of PrimPol as a leading strand repriming enzyme and describe the mechanisms responsible for recruiting and regulating the enzyme during this process. This review provides an overview and update of the current PrimPol literature, as well as highlighting unanswered questions and potential future avenues of investigation. Full article