Sensing of DNA modifications by pAgo proteins in vitro

Many prokaryotic Argonaute (pAgo) proteins act as programmable nucleases that use small guide DNAs for recognition and cleavage of complementary target DNA. Recent studies suggested that pAgos participate in cell defense against invader DNA and may also be involved in other genetic processes, including DNA replication and repair. The ability of pAgos to recognize specific targets potentially make them an invaluable tool for DNA manipulations. Here, we demonstrate that DNA-guided DNA-targeting pAgo nucleases from three bacterial species, DloAgo from Dorea longicatena, CbAgo from Clostridium butyricum and KmAgo from Kurthia massiliensis, can sense site-specific modifications in the target DNA, including 8-oxoguanine, thymine glycol, ethenoadenine and pyrimidine dimers. The effects of DNA modifications on the activity of pAgos strongly depend on their positions relative to the site of cleavage and are comparable to or exceed the effects of guide-target mismatches at corresponding positions. For all tested pAgos, the strongest effects are observed when DNA lesions are located at the cleavage position. The results demonstrate that DNA cleavage by pAgos is strongly affected by DNA modifications, thus making possible their use as sensors of DNA damage.