Cyanobacterial Argonautes and Cas4 family nucleases cooperate to interfere with invading DNA
Pilar Bobadilla Ugarte, Stefanie Halter, Sumanth Mutte, Clint Heijstek, Théophile Niault, Ilya M. Terenin, Patrick Barendse, Balwina Koopal, Mark Roosjen, Sjef Boeren, Vasili Hauryliuk, Martin Jínek, Adrie H. Westphal, Daan C. Swarts
Abstract
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.