Cyanobacterial Argonautes and Cas4 family nucleases cooperate to interfere with invading DNA.

Prokaryotic Argonaute proteins (pAgos) from the long-A clade are stand-alone immune systems that use small interfering DNA (siDNA) guides to recognize and cleave invading plasmid and virus DNA. Certain long-A pAgos are co-encoded with accessory proteins with unknown functions. Here, we show that cyanobacterial long-A pAgos act in conjunction with Argonaute-associated Cas4 family enzyme 1 (ACE1). Structural and biochemical analyses reveal that ACE1-associated pAgos mediate siDNA-guided DNA interference, akin to stand-alone pAgos. ACE1 is structurally homologous to the nuclease domain of bacterial DNA repair complexes and acts as a single-stranded DNA endonuclease that processes siDNA guides. pAgo and ACE1 form a heterodimeric long-A pAgo-ACE1 (APACE1) complex, which modulates ACE1 activity. Although ACE1-associated pAgos alone interfere with plasmids and bacteriophages, plasmid interference is boosted when pAgo and ACE1 are co-expressed. Our study reveals that pAgo-mediated immunity is enhanced by accessory proteins and broadens our mechanistic understanding of how pAgo systems interfere with invading DNA.