RESUMO
CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.
