Motif B and Lys297 are required to stabilize 3¿ incoming deoxynucleotides by human PrimPol

Trabajo presentado en el 6th DNA Polymerases meeting, celebrado en Stockholm (Suecia) del 22 al 25 de mayo de 2022.Human PrimPol is a DNA primase/polymerase that enables progression of stalled replication forks by synthesizing DNA primers after blocking lesions or structures in the DNA template. PrimPol belongs to the Archaeo-Eukaryotic primase superfamily (AEPs) which contains a catalytic core with three conserved motifs: A, B and C. Inspection of the crystal structure of human PrimPol confirmed that Motifs A and C harbour the acidic residues acting as metal ligands for catalysis (Asp114, Glu116, Asp280 in human PrimPol), whereas Motif B includes several residues as candidates to interact with the 3¿ incoming deoxynucleotide (Ser160, Lys165, Ser167 and His169); three of them are highly conserved in motif B (KxSxH) of PrimPols, as determined by multiple amino acid sequence alignment. Additionally, other putative nucleotide ligands are situated in Motif C (Arg288, Asn289, Arg291), or in a close proximity (Lys297 and Lys300). Whereas Lys297 is almost invariant in the whole AEP superfamily, Arg288, Arg291 and Lys300 are specially conserved in eukaryotic PrimPols. Although Motifs A and C have been deeply studied, little information is available about the specific function of motif B, and about the contribution of individual residues for binding the 3¿ incoming nucleotide. By site-directed mutagenesis, we confirmed that His169 was essential for both primase/polymerase activities of PrimPol, whereas Ser167 and Lys297 were crucial for dimer synthesis when priming, but not for subsequent dNTP incorporation on primers. On the other hand, elimination of Lys165 did not affect primase function but affected primer-template realignments required for PrimPol TLS-activities. Finally, Lys300 was irrelevant for priming, but contributed to PrimPol polymerase activity. Collectively, these results strongly suggest that individual 3¿ incoming nucleotide ligands have a different impact in the stabilization of the nucleotide. It is likely that specific combinations of these residues could be required to provide the maximal strength of interaction with the nucleotide during dimer formation and translesion synthesis by PrimPol