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To stabilize or not to stabilize RNA - that is still the question

Updated: Jan 24, 2022

Publishing venue: Science Advances - 11 Jun 2021: Vol. 7, no. 24, eabf1771


Link: https://advances.sciencemag.org/content/7/24/eabf1771


Author(s) and Title: Chandramouly, G., et al. Polθ reverse transcribes RNA and promotes RNA-templated DNA repair


In the June 11th issue of Science Advances, G. Chandramouly, et al. published an excellent article about the promiscuous reverse transcriptase function of DNA polymerase theta (Polθ; EC no. 2.7.7.7) with special emphasis on its potential DNA transforming effect also involved in cancer formation (1). A few days later the aminocoumarin antibiotic novobiocin, as the first-in-class Polθ inhibitor, was reported that selectively targets homologous-recombination-deficient tumor cells with therapeutic implications (2). The authors significantly contribute herein to elucidate the catalytic activity of Polθ, which appears similar to other polymerases in their dependence on multiple hydrogen bonds and transfers; in this case that with the template’s ribose 2′-hydroxyl groups. Most importantly the authors elucidate the previously inconclusive role of water molecules to control the rate constant for nucleotide incorporation in the pre-steady state (3), as proton-inventory experiments have consistently revealed two protons being transferred during the rate-limiting transition state of the reaction, which is also described for human immunodeficiency virus (HIV) reverse transcriptase. A loop shift in the palm subdomain of Polθ is now apparent, suggesting a specific ribose 2′-hydroxyl interaction with main-chain domain carbonyl groups mediated most likely through a water molecule.

This indicates a prominent role of water to control the rate constant for nucleotide incorporation in the pre-steady state that has previously been only hinted when a solvent (D2O/H2O) deuterium kinetic isotope effect of 3 ± 0.5 (Mg2+) and 7 ± 2 (Mn2+) was observed for AMP incorporation in the pre-steady-state at pH 7.5 for nucleotide incorporation (3). Unaffected kinetic properties of AMP incorporation in the steady state ruled out conformational perturbations as the cause of the observed water solvent-based isotope effects by all target RNA and DNA polymerases (4); indicating the importance of undisrupted proton transfers from water, in the absence of deuterium, to reach steady state substrate/product relationship kinetics during copy DNA production.

The above discoveries are important as A) RNA and DNA polymerases are inducible by nucleic acid template architectures of various pathogenic and/or endogenous origins (5), and B) many, if not all, RNA (6) and DNA (7) template architectures harbor significant deuterium-related conformational stability and resistance against degradation. For example, random integration (RI) frequently occurs at double-strand breaks (DSBs) in the genome, whereby ionizing radiation and viral interference with deuterium-meditated genomic stability readily increase the frequency of integration. This warrants further exploration in vivo to determine if deutenomics, i.e. the study of deuterium discrimination, compartmentalization and fractionation in living cells, implies to radiation and/or gene therapy risk assessment.

It seems that the RNA template and DNA break dependence of Polθ also deserve a closer look in order to decipher its cell transforming effect by transcribing stable RNA templates in experiments with mammalian cells. The physiological inherent instability of ribonucleic acid templates is the result of a spontaneous cleavage of its phosphodiester linkages via intramolecular transesterification reactions (8). Under a broad range of physiological conditions, the rapid and spontaneous breakdown of RNA occurs to preserve genetic integrity by preventing the reverse transcriptase action of Polθ at its template substrate level in mammalian cells. Additional mechanisms that rapidly dismantle and/or modify RNA molecules to enhance their chemical instability include nucleases, involved in cleaving the phosphodiester bonds between nucleotides, and ribozymes, involved in splicing. Due to the above facts, in order for the Polθ enzyme to reverse transcribe RNA sequences and fragments stable ribonucleotide templates are required, which are available in deuterated water loaded cancer cells either by glycolytic activity with semi-deuterated metabolic water formation (9, 10) or by mitochondrial damage that compromises deuterium-depleting proton exchange reactions in the Krebs-Szent-Györgyi cycle during the Warburg metabolism (11). A recently published report corroborates the prime role of intracellular water in the transformation between a normal and cancerous phenotype of human cells with aneuploidy (12). This is by demonstrating through accurate quasi-elastic neutron scattering experiments that a change in the dynamic flexibility of structured water correlates with the existence of pathology. Apparently, chemically modified stable DNA and RNA templates for genetically delivered therapies, made with heavy water (13), as well as deuterium-rich nutrients with epidemiological scales (14), likely all trigger conformational changes in Polθ’s palm subdomain, catalytic sites and function, whereby Polθ’s promiscuous water and proton dependence may adversely affect cancer incidence and outcomes. The ternary structure of Polθ on a DNA/RNA primer-template, as characterized by interactions with deuterium prone 2′-hydroxyl and 3’- ribose hydrogen bonds, suppress Polθ template misalignment errors and thus potentially contribute to its higher fidelity on RNA (1).

Future genetic interventions and therapies should consider Polθ as a substrate and solvent-dependent reverse transcriptase, targeting stable RNA adducts, with significant implications in translational and clinical medicine.


REFERENCES


1. G. Chandramouly, J. Zhao, S. McDevitt, T. Rusanov, T. Hoang, N. Borisonni, T. Treddinick, F. W. Lopezcolorado, T. Kent, L. A. Siddique, J. Mallon, J. Huhn, Z. Shoda, E. Kashkina, A. Brambati, J. M. Stark, X. S. Chen, R. T. Pomerantz. Polθ reverse transcribes RNA and promotes RNA-templated DNA repair. Sci. Adv. (2021) 7, eabf1771, doi: 10.1126/sciadv.abf1771


2. J. Zhou, C. Gelot, C. Pantelidou, et al. A first-in-class polymerase theta inhibitor selectively targets homologous-recombination-deficient tumors. Nat. Cancer. (2021). https://doi.org/10.1038/s43018-021-00203-x


3. C. Castro, E. Smidansky, K. R. Maksimchuk, J. J. Arnold, V. S. Korneeva, M. Götte, W. Konigsberg, C. E. Cameron. Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. Proc. Natl. Acad. Sci. U S A. (2007) 104, 4267-72. doi: 10.1073/pnas.0608952104


4. C. Castro, E. D. Smidansky, J. J. Arnold, K. R. Maksimchuk, I. Moustafa, A. Uchida, M. Götte, W. Konigsberg, C. E. Cameron. Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat. Struct. Mol. Biol. (2009) 16(2), 212-8. doi: 10.1038/nsmb.1540


5. J. Rentergent, M. D Driscoll, S. Hay. 6. Time Course Analysis of Enzyme-Catalyzed DNA Polymerization. Biochemistry (2016) 55, 5622-5634. doi: 10.1021/acs.biochem.6b00442


6. B. Balasubramanian, W. K. Pogozelski, T. D. Tullius. DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc. Natl. Acad. Sci. U S A. (1998) 95(17): 9738-43. doi: 10.1073/pnas.95.17.9738


7. B. Chen, E. R. Jamieson, T. D. Tullius. A general synthesis of specifically deuterated nucleotides for studies of DNA and RNA. Bioorg. Med. Chem. Lett. (2002) 12(21), 3093-6. doi: 10.1016/s0960-894x(02)00650-9


8. G. A. Soukup, R. R. Breaker. Relationship between internucleotide linkage geometry and the stability of RNA. RNA (1999) 5(10), 1308-25. doi: 10.1017/s1355838299990891


9. R. Mahar, P. L. Donabedian, M. E. Merritt. HDO production from [2H7]glucose Quantitatively Identifies Warburg Metabolism. Sci. Rep. (2020) 10(1), 8885. doi: 10.1038/s41598-020-65839-8


10. L. G. Boros, T. Q. Collins, E. A. Boros, F. Lantos, G. Somlyai. Deuterium and metabolic water matter – what this means biochemically and clinically. Sci. Adv. (2018) 4(8): eaat7314. https://advances.sciencemag.org/content/4/8/eaat7314/tab-e-letters


11. L. G. Boros, D. P. D’Agostino, H. E. Katz, J. P. Roth, E. J. Meuillet, G. Somlyai. Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in the tricarboxylic acid substrate cycle. Med. Hypotheses. (2016) 87: 69-74, doi: 10.1016/j.mehy.2015.11.016


12. M. P. M. Marques, A. L. M. Batista de Carvalho, A. P. Mamede, A. Dopplapudi, V. García Sakai, L. A. E. Batista de Carvalho. Role of intracellular water in the normal-to-cancer transition in human cells-insights from quasi-elastic neutron scattering. Struct. Dyn. (2020) 7(5), 054701. doi: 10.1063/4.0000021. eCollection


13. A. Sen, V. Balamurugan, K. K. Rajak, S. Chakravarti, V. Bhanuprakash, R. K. Singh. Role of heavy water in biological sciences with an emphasis on thermostabilization of vaccines. Expert. Rev. Vaccines. (2009) 8, 1587-602, doi: 10.1586/erv.09.105


14. L. G. Boros, T. Q. Collins, G. Somlyai. What to eat or what not to eat ‐ that is still the question. Neuro. Oncol. (2017) 19(4): 595-596, doi: 10.1093/neuonc/now284


from: SciAdv-eletter@highwire.org

to: "László G. Boros" <contact@laszlogboros.com>

date: Jun 26, 2021, 9:47 AM

subject: Thank you for your eLetter response to Science Advances

mailed-by: auto-drupal-prod-web38.highwire.org

Keywords: Polymerase Theta; Ribonucleic acid; RNA; Deoxyribonucleic acid; DNA; Medical Deutenomics; MDeutenomics; Stable RNA; Reverse transcriptase, Peroxisomes


Full list of authors:

László G. Boros, Professor of Pediatrics/M.D.,

Harbor-UCLA Medical Center and Chief Scientist, Deutenomics Science Institute, SIDMAP, LLC, Culver city, CA, USA - Correspondence: contact@laszlogboros.com

ORCID - https://orcid.org/0000-0003-1211-9933


Other Contributors:

Tjaart P. J. Krüger, Professor of Physics/Ph.D.,

Department of Physics and the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa

Email: tjaart.kruger@up.ac.za

ORCID - https://orcid.org/0000-0002-0801-6512


Tamás Letoha, Chief Executive Officer/M.D., Ph.D.,

Pharmacoidea Ltd, Szeged, Hungary

Email: tamas.letoha@pharmacoidea.eu

ORCID - https://orcid.org/0000-0002-6035-4009


Jack A. Tuszynski, Professor/Ph.D.,

Department of Physics, Biomedical Engineering and Oncology, The Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada

Email: jackt@ualberta.ca

ORCID - https://orcid.org/0000-0001-9976-0429


Petra Davelaar Dorfsman*, Integrative Deutenomics/N.D. C.N.S. I.F.M.C.P.,

Deutenomics Science Institute, Balassagyarmat, Hungary

Email: info@drpetrad.com

ORCID - https://orcid.org/0000-0002-9697-0110


James C. Lech*, Chair and Scientific Advisor/M.S.,

World Health Organization, EMF Project, South Africa, National Research Foundation, South Africa and Faculty of Science, Vrije Universiteit Amsterdam, The Netherlands

Email: james.lech@vu.nl

ORCID - https://orcid.org/0000-0001-9919-0120

(*These authors contributed equally to the eLetter)


SciAdv-eletter@highwire.org - Jun 26, 2021, 9:47 AM - The eLetter was submitted on 26 Jun 2021


Article (citation): Chandramouly, Gurushankar et al "Polθ reverse transcribes RNA and promotes RNA-templated DNA repair." Science Advances 7.24 (2021): eabf1771. Web. 26 June. 2021.



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