Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute

Abstract

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA

“…Similar to previously published structures of RsAgo (Miyoshi et al, 2016) and TtAgo (Sheng et al, 2014; Swarts et al, 2014a, 2017; Wang et al, 2009), the 5′-phosphate of the guide strand is positioned within the MID pocket and anchored in place through a network of direct hydrogen bonds from amino acid side chains originating from the MID (Tyr463, Lys467, Gln478, and Lys506) domain (Figures 3A and 3B). Indeed, mutation of Tyr463 and Lys467 residues make RsAgo unable to bind RNA guides in vivo (Olovnikov et al, 2013) and in vitro (Miyoshi et al, 2016).…”

“…Similarly, sequence-specific recognition of the 5′-base in the guide strand were shown for several eukaryotic Agos (Boland et al, 2010; Frank et al, 2010; Matsumoto et al, 2016; Schirle and MacRae, 2012; Wang et al, 2009; Mi et al, 2008). On the contrary, TtAgo was proposed to have little specificity for the 5′-base binding (Swarts et al, 2014a, 2017). …”

“…By contrast, TtAgo preferentially binds G1′ in the target strand, which is also stabilized by specific hydrogen bonding (Sheng et al, 2014; Swarts et al, 2017). Thus, the ability to recognize the target nucleotide at position 1′ may be an ancient feature of Ago proteins present in the common ancestor of prokaryotic and eukaryotic lineages.…”

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit

“…The pre-crRNA is cleaved within the repeat region, which adopts a pseudoknot structure (Swarts et al, 2017; Yamano et al, 2016). In the structure of Cas12a bound to a pre-crRNA, the scissile phosphate is directly positioned between the sidechains of highly-conserved histidine and lysine residues within the WED domain (Swarts et al, 2017).…”

“…The pre-crRNA is cleaved within the repeat region, which adopts a pseudoknot structure (Swarts et al, 2017; Yamano et al, 2016). In the structure of Cas12a bound to a pre-crRNA, the scissile phosphate is directly positioned between the sidechains of highly-conserved histidine and lysine residues within the WED domain (Swarts et al, 2017). Although early studies suggested that pre-crRNA cleavage requires divalent metal ions (Fonfara et al, 2016), subsequent studies showed that Mg 2+ ions help stabilize Cas12a binding to the RNA but are not required for catalysis (Swarts et al, 2017).…”