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

Area of Science:

• Molecular Biology
• Enzymology
• DNA Replication

Background:

• PrimPol is a DNA primase/polymerase in the Archaeo-Eukaryotic Primase (AEP) superfamily.
• It facilitates stalled replication fork progression by synthesizing DNA primers.
• PrimPol's active site involves three conserved motifs (A, B, C) and additional residues.

Purpose of the Study:

• To elucidate the specific roles of key residues in human PrimPol (HsPrimPol) during DNA primer synthesis.
• To understand how individual ligands contribute to deoxynucleotide (dNTP) binding and stabilization.
• To investigate the function of PrimPol in replication fork progression and damage avoidance.

Main Methods:

• Site-directed mutagenesis of specific PrimPol residues (His169, Ser167, Lys297, Lys165, Lys300).
• Assays to measure primase and polymerase activities.
• Analysis of deoxynucleotide (dNTP) binding and stabilization.
• Evaluation of PrimPol's role in primer-template realignment for damage avoidance.

Main Results:

• His169 is essential for dNTP binding and both primase/polymerase activities.
• Ser167 and Lys297 are critical for primer synthesis initiation but not elongation.
• Lys165 is dispensable for primase function but required for damage avoidance.
• Lys300 acts as an additional residue for stabilizing incoming dNTPs.

Conclusions:

• Individual ligands in HsPrimPol modulate 3' incoming dNTP stabilization.
• These modulations optimize DNA primer synthesis efficiency during initiation and maturation.
• HsPrimPol's distinct residues contribute to both replication restart and DNA damage tolerance mechanisms.