Initial primer synthesis of a DNA primase monitored by real-time NMR spectroscopy

Type
01A - Journal article
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Journal of the American Chemical Society
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Volume
146
Issue / Number
14
Pages / Duration
9583-9596
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American Chemical Society
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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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0002-7863
1520-5126
Language
English
Created during FHNW affiliation
Yes
Strategic action fields FHNW
Publication status
Unpublished
Review
Peer review of the complete publication
Open access category
Hybrid
License
'https://creativecommons.org/licenses/by/4.0/'
Citation
Wu, P., Zehnder, J., Schröder, N., Blümmel, P. E. W., Salmon, L., Damberger, Fred. F., Lipps, G., Allain, F. H.-T., & Wiegand, T. (2024). Initial primer synthesis of a DNA primase monitored by real-time NMR spectroscopy. Journal of the American Chemical Society, 146(14), 9583–9596. https://doi.org/10.1021/jacs.3c11836