CRISPR-Cas induced self-targeting identifies key players in archaeal microhomology-mediated end joining.

Abstract

DNA repair processes are the foundation for genome integrity and survival, especially in extreme environments where DNA damage occurs more frequently and where archaea are found. Nevertheless, first-hand experimental information on repair pathways in archaea is scarce, and assignment of repair proteins is currently largely based on homology. We showed previously that DNA lesions induced by clustered regularly interspaced short palindromic repeats Cas (CRISPR-Cas) self-targeting are repaired by microhomology-mediated end joining (MMEJ). To identify proteins involved in the archaeal MMEJ pathway, we used deletion strains devoid of proteins assigned to the key steps of MMEJ, to examine changes in the repair outcome. In addition, we used aphidicolin to inhibit the activity of the essential PolB1 protein. For the first time, we were thereby able to experimentally identify proteins involved in this repair pathway in the euryarchaeal model organism Haloferax volcanii. This study confirms that Mre11, Rad50, Fen1, PolB1, LigA, and LigN take part in MMEJ, as previously inferred. In addition, we show that Cas1 and Hel308a are also involved in the MMEJ pathway.