Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute

• Published in: Molecular cell Vol. 65; no. 6; pp. 985 - 998.e6
• Main Authors: Swarts, Daan C., Szczepaniak, Malwina, Sheng, Gang, Chandradoss, Stanley D., Zhu, Yifan, Timmers, Elizabeth M., Zhang, Yong, Zhao, Hongtu, Lou, Jizhong, Wang, Yanli, Joo, Chirlmin, van der Oost, John
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.03.2017
• Subjects: Ago; Argonaute Proteins - chemistry; Argonaute Proteins - genetics; Argonaute Proteins - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Deoxycytidine - metabolism; Deoxyguanosine - metabolism; DNA chopping; DNA, Antisense - chemistry; DNA, Antisense - genetics; DNA, Antisense - metabolism; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Fluorescence Resonance Energy Transfer; Guide generation; Guide loading; Molecular Dynamics Simulation; Nucleic Acid Conformation; PAgo; Prokaryotic argonaute; Protein Binding; Protein Conformation; RNA interference; SiDNA; Single Molecule Imaging; Small interfering DNA; Structure-Activity Relationship; Thermus thermophilus - enzymology; Thermus thermophilus - genetics; TtAgo; RNA interference; guide loading; small interfering DNA; siDNA; DNA chopping; ago; pAgo; prokaryotic argonaute; TtAgo; guide generation
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2017.01.033

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. [Display omitted] •Guide-free TtAgo preferentially binds dsDNA over single-stranded DNA•Guide-free TtAgo can generate small interfering DNAs by chopping (degrading) dsDNA•TtAgo does not have preference for a specific 5′ end nucleotide on the guide strand•TtAgo preferentially binds a deoxyguanosine on the passenger strand Swarts et al. demonstrate that, in absence of a guide, the Argonaute protein from Thermus thermophilus chops (degrades) double-stranded DNA. Chopped DNA is sequence-specifically bound by TtAgo, which results in loading of DNA guides with a 5′ end deoxycytidine. The TtAgo-guide complex can subsequently bind and cleave cognate DNA targets.