Open Readings 2023 • P3-40
ACTIVITY AND CRYSTALLIZATION OF TYPE III CRISPR-CAS ASSOCIATED RING NUCLEASE
Danas Klimavičius1, Dominik Rafalski2, Matthias Bochtler2, 3, Gintautas Tamulaitis1, 4
1 Institute of Biotechnology, Life Sciences Center, Vilnius University, Lithuania
2 International Institute of Molecular and Cell Biology, Poland
3 Institute of Biochemistry and Biophysics PAS, Poland
4 Life Sciences Center European Molecular Biology Laboratory Partnership Institute, Vilnius University, Lithuania
[email protected]
Type III CRISPR-Cas systems employ Csm or Cmr ribonucleoprotein complexes to cleave RNA [1] and DNA [2] to confer immunity against viruses and foreign plasmids. After recognition of target RNA Csm/Cmr complex also synthesizes 3-6 nt cyclic oligoadenylates (cOA) from ATP [3]. cA4 or cA6 allosterically activates CARF domain-containing accessory proteins. Bioinformatic analysis revealed that in prokaryotic genomes CARF domain could be fused with different putative DNases, RNases, adenosine deaminase and other domains [4]. Together with Csm/Cmr these auxiliary effectors provide immunity to the host. However, they could be toxic to a cell and result a dormancy or an altruistic cell death. To avoid host damage, the cOA concentration is tightly regulated by (i) cOA synthesis that is controlled through target RNA degradation by Csm complex [2], (ii) cOA degradation by specialized enzymes called ring nucleases [5,6].
Several uncharacterized CRISPR-associated genes were hypothesized to possess ring nuclease activity [4]. In this study we present results to demonstate that Csx20 family proteins act as ring nucleases in vitro and in vivo. We also share results of our crystallization attempts in order to determine nuclease structure and mechanism.
[1] G. Tamulaitis, M. Kazlauskiene, E. Manakova, C. Venclovas, A. O. Nwokeoji, M. J. Dickman, P. Horvath, V. Siksnys, 2014, Mol. Cell, 4, p. 506-517.
[2] M. Kazlauskiene, G. Tamulaitis, G. Kostiuk, Č. Venclovas, V. Siksnys, 2016, Mol. Cell, 2, p. 295-306.
[3] M. Kazlauskiene, G. Kostiuk, Č. Venclovas, G. Tamulaitis, V. Siksnys, 2017, Science 357(6351), p. 605-609.
[4] K. S. Makarova, A. Timinskas, Y. Wolf, A. B. Gussow, V. Siksnys, Č. Venclovas, E. V. Koonin, 2020, Nucleic Acids Res., 16, p. 8828-8847.
[5] J. S. Athukoralage, C. Rouillon, S. Graham, S. Grüschow, M. F. White, 2018, Nature, 7726, p. 277-280.
[6] D. Smalakyte, M. Kazlauskiene, J. Havelund, A. Ruksenaite, A. Rimaite, G. Tamulaitiene, N. J. Færgeman, G. Tamulaitis, V. Siksnys, 2020, Nucleic Acids Res., 16, p. 9204-9217.
