Abstract
Cas3 is a signature protein of the type I CRISPR-Cas systems and typically contains HD phosphohydrolase and Superfamily 2 (SF2) helicase domains. In the type I CRISPR-Cas systems Cas3 functions as a slicer that provides foreign DNA degradation. Biochemical analysis indicate that Cas3 of the Streptococcus thermophilus DGCC7710 (St-Cas3) CRISPR4 system is a single-stranded DNA nuclease which also possesses a single-stranded DNA-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes in 3′ to 5′ direction. The interplay between the nuclease and ATPase/helicase activities of St-Cas3 results in DNA degradation. Here, we describe assays for monitoring of St-Cas3 nuclease, ATPase and helicase activities in a stand-alone form and in the presence of the Cascade ribonucleoprotein complex. These assays can be easily adapted for biochemical analysis of Cas3 proteins from different microorganisms.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712. doi:10.1126/science.1138140
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170, 10.1126/science.1179555
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338. doi:10.1038/nature10886
Gasiunas G, Sinkunas T, Siksnys V (2013) Molecular mechanisms of CRISPR-mediated microbial immunity. Cell Mol Life Sci. doi:10.1007/s00018-013-1438-6
Makarova KS, Haft DH, Barrangou R et al (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467–477. doi:10.1038/nrmicro2577
Sinkunas T, Gasiunas G, Fremaux C et al (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 30:1335–1342. doi:10.1038/emboj.2011.41
Sinkunas T, Gasiunas G, Waghmare SP et al (2013) In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. EMBO J 32:385–394. doi:10.1038/emboj.2012.352
Haft DH, Selengut J, Mongodin EF, Nelson KE (2005) A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 1:e60. doi:10.1371/journal.pcbi.0010060
Makarova KS, Grishin NV, Shabalina SA et al (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7. doi:10.1186/1745-6150-1-7
Westra ER, van Erp PBG, Künne T et al (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 46:595–605. doi:10.1016/j.molcel.2012.03.018
Mulepati S, Bailey S (2011) Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J Biol Chem 286:31896–31903. doi:10.1074/jbc.M111.270017
Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A 74:560–564
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560
Acknowledgement
We thank Rodolphe Barrangou and Philippe Horvath for discussions. The work on CRISPR systems in Siksnys’s laboratory was funded by the European Social Fund under Global Grant measures.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sinkunas, T., Gasiunas, G., Siksnys, V. (2015). Cas3 Nuclease–Helicase Activity Assays. In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2687-9_18
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2686-2
Online ISBN: 978-1-4939-2687-9
eBook Packages: Springer Protocols