biostudies-literatureBobadilla Ugarte PEuropean Molecular Biology OrganizationEuropean Research CouncilDutch Research Council (NWO)National Council on Science and Technology1920-1937.e10https://www.ebi.ac.uk/biostudies/studies/S-EPMC12094033biostudies-literatureUnknown85(10)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.Molecular cellCyanobacterial Argonautes and Cas4 family nucleases cooperate to interfere with invading DNA.PMC12094033016.Veni.192.072948783Bobadilla Ugarte PHauryliuk VBoeren SWestphal AHMutte SKBarendse PHalter SHeijstek CTerenin ISwarts DCJinek MNiault TRoosjen MKoopal BfalseCyanobacterial 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.2025-01-01T00:00:00Z2025 May2026-06-01T10:22:16.588Z2026-04-08T11:22:02.708ZS-EPMC120940334028837410.1016/j.molcel.2025.03.025