3'dNTP Binding Is Modulated during Primer Synthesis and Translesion by Human PrimPol.

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 ( Hs PrimPol) harbor the catalytic residues (Asp 114 , Glu 116 , Asp 280 ) acting as metal ligands, whereas motif B includes highly conserved residues (Lys 165 , Ser 167 and His 169 ), which are postulated to stabilize 3' incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys 297 , almost invariant in the whole AEP superfamily, and Lys 300 , specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His 169 is absolutely essential for 3'dNTP binding and, hence, for both primase and polymerase activities of Hs PrimPol, whereas Ser 167 and Lys 297 are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys 165 does not affect the overall primase function; however, it is required for damage avoidance via primer-template realignments. Finally, Lys 300 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.