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PROOFREADING DOMAIN FACILITATES SPACER INTEGRATION IN CRISPR-CAS – AN ADAPTIVE PROKARYOTIC IMMUNITY Gediminas Drabavičius, Arūnas Šilanskas, Tomas Šinkūnas, Giedrius Gasiūnas Institute of Biotechnology, Vilnius University, Lithuania [email protected] Bacteria and archaea are constantly assailed by viral pathogens. Like higher organisms, they have evolved defense systems, most of which could be likened to innate immunity. However, some microorganisms have an adaptive system called CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats). This system works in three stages: (I) Adaptation, where pieces of destroyed viral DNA are incorporated into genomic CRISPR loci as spacers between identical repeats; (II) crRNA Maturation, where CRISPR locus and Cas genes are transcribed and effector complexes assemble; (III) Interference, where effector complexes, which patrol the cell find a pathogen which has complementary DNA to a spacer from CRISPR locus and destroys it. Between different prokaryotes, a wide variety of CRISPR-Cas systems exist. They are subdivided into Classes 1 and 2. Class 1 is composed of CRISPR-Cas systems which has an effector complex composed of several proteins, whereas Class 2 exhibit effector complexes of a single protein, most famous of which is Cas9 [1, 2]. These Classes are further divided into Types and Subtypes. Despite this great variety, the adaptation (spacer acquisition) is achieved by similar means in all of them. All systems have highly conserved Cas1 and Cas2 proteins responsible for the immunization of bacteria or archaea. In this study, we investigated Cas1 and Cas2 proteins from Streptococcus thermophilus strain DGCC7710. This bacterium has 4 different CRISPR-Cas systems and is very important for milk industry. Furthermore, it was the first microorganism in which the function of CRISPR-Cas systems, as an anti-viral defense mechanism, was demonstrated [3]. We chose CRISPR4-Cas system. It is unique in that its Cas2 protein is fused to DnaQ domain, which is an exonuclease domain homologous to ϵ subunit of Escherichia coli DNA polymerase. ϵ subunit is responsible for proofreading activity [4, 5]. Although several organisms exhibit Cas2-DnaQ fusion, its function in integration remained obscure. In this study, we set out to find out the function and a role of the DnaQ domain in CRISPR4-Cas adaptation. [1] Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas systems." Nature Reviews Microbiology (2015). [2] Mohanraju, Prarthana, et al. "Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems." Science 353.6299 (2016). [3] Barrangou, Rodolphe, et al. "CRISPR provides acquired resistance against viruses in prokaryotes." Science 315.5819 (2007): 1709-1712. [4] Huang, Yiping, Dan K. Braithwaite, and Junetsu Ito. "Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria." FEBS letters 400.1 (1997): 94-98. [5] Hamdan, Samir, et al. "Hydrolysis of the 5 ‘-p-Nitrophenyl Ester of TMP by the Proofreading Exonuclease (ε) Subunit of Escherichia coli DNA Polymerase III." Biochemistry 41.16 (2002): 5266-5275.
