Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute
Daan C. Swarts (Wageningen University & Research)
Malwina Szczepaniak (TU Delft - BN/Chirlmin Joo Lab)
Gang Sheng (Chinese Academy of Sciences)
Stanley D. Chandradoss (TU Delft - BN/Chirlmin Joo Lab)
Yifan Zhu (Wageningen University & Research)
Elizabeth M. Timmers (Wageningen University & Research)
Yong Zhang (Chinese Academy of Sciences)
Hongtu Zhao (Chinese Academy of Sciences)
Jizhong Lou (Chinese Academy of Sciences)
Yanli Wang (Chinese Academy of Sciences)
Chirlmin Joo (TU Delft - BN/Chirlmin Joo Lab)
John van der Oost (Wageningen University & Research)
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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.
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