Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• PrimPol is essential for DNA replication priming in both nuclear and mitochondrial DNA.
• Dideoxynucleotides (ddNTPs) are chain terminators used to block DNA replication, particularly in mitochondria via Polγ.
• Understanding PrimPol's nucleotide selectivity is key to its role in DNA replication and repair.

Purpose of the Study:

• To investigate human PrimPol's discrimination against dideoxynucleotides (ddNTPs) during DNA primer synthesis and elongation.
• To elucidate the molecular mechanism underlying PrimPol's selectivity for deoxynucleotides (dNTPs) over ddNTPs.
• To assess the impact of ddNTP incorporation on PrimPol's primase and polymerase activities.

Main Methods:

• Biochemical assays measuring PrimPol's incorporation of dNTPs versus ddNTPs.
• Analysis of PrimPol's *de novo* primer synthesis and elongation across lesions.
• Crystal structure determination of human PrimPol to identify key residues involved in nucleotide selectivity.

Main Results:

• Human PrimPol exhibits strong discrimination against ddNTPs during *de novo* primer synthesis and elongation, especially at the initial dimer formation.
• PrimPol incorporates ddNTPs with significantly lower efficiency than dNTPs, even with activating manganese ions.
• The crystal structure reveals Arg291 is critical for discriminating against ddNTPs by interacting with the 3'-OH group, absent in ddNTPs.

Conclusions:

• PrimPol's selectivity against ddNTPs prevents premature primer termination, ensuring functional primer synthesis for DNA replication.
• The Arg291 residue in PrimPol is crucial for its dNTP/ddNTP selectivity, contributing to its primase and polymerase activities.
• This discrimination mechanism safeguards DNA replication integrity, particularly in the context of mitochondrial DNA maintenance.