Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy

TL;DR

Scientists used NMR to study how a DNA primase enzyme starts DNA replication by forming a short primer.

Contribution

The study introduces real-time NMR methods to observe the initial steps of primer synthesis by an archaeal primase.

Findings

The helix-bundle domain positions substrates before transferring them to the catalytic domain.

Solid-state NMR can monitor dinucleotide formation kinetics similarly to solution-state NMR.

Conformational changes in the enzyme during primer synthesis are detected using high-frequency NMR.

Abstract

Primases are crucial
enzymes for DNA replication, as they synthesize
a short primer required for initiating DNA replication. We herein
present time-resolved nuclear magnetic resonance (NMR) spectroscopy
in solution and in the solid state to study the initial dinucleotide
formation reaction of archaeal pRN1 primase. Our findings show that
the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates
and then hands them over to the catalytic domain to initiate the reaction.
By using nucleotide triphosphate analogues, the reaction is substantially
slowed down, allowing us to study the initial dinucleotide formation
in real time. We show that the sedimented protein–DNA complex
remains active in the solid-state NMR rotor and that time-resolved 31P-detected cross-polarization experiments allow monitoring
the kinetics of dinucleotide formation. The kinetics in the sedimented
protein sample are comparable to those determined by solution-state
NMR. Protein conformational changes during primer synthesis are observed
in time-resolved 1H-detected experiments at fast magic-angle
spinning frequencies (100 kHz). A significant number of spectral changes
cluster in the HBD pointing to the importance of the HBD for positioning
the nucleotides and the dinucleotide.

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