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-conserv...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-12-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/25/1/51 |
| Summary: | 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 (<i>Hs</i>PrimPol) harbor the catalytic residues (Asp<sup>114</sup>, Glu<sup>116</sup>, Asp<sup>280</sup>) acting as metal ligands, whereas motif B includes highly conserved residues (Lys<sup>165</sup>, Ser<sup>167</sup> and His<sup>169</sup>), which are postulated to stabilize 3′ incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys<sup>297</sup>, almost invariant in the whole AEP superfamily, and Lys<sup>300</sup>, specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His<sup>169</sup> is absolutely essential for 3′dNTP binding and, hence, for both primase and polymerase activities of <i>Hs</i>PrimPol, whereas Ser<sup>167</sup> and Lys<sup>297</sup> are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys<sup>165</sup> does not affect the overall primase function; however, it is required for damage avoidance via primer–template realignments. Finally, Lys<sup>300</sup> 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. |
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| ISSN: | 1661-6596 1422-0067 |