@article{8adbe4fec2b445bc876fc1374c2d8494,
title = "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.",
keywords = "DNA chopping, RNA interference, TtAgo, ago, guide generation, guide loading, pAgo, prokaryotic argonaute, siDNA, small interfering DNA",
author = "Swarts, {Daan C.} and Malwina Szczepaniak and Gang Sheng and Chandradoss, {Stanley D.} and Yifan Zhu and Timmers, {Elizabeth M.} and Yong Zhang and Hongtu Zhao and Jizhong Lou and Yanli Wang and Chirlmin Joo and {van der Oost}, John",
note = "Funding Information: The authors thank Martin Jinek for permission to use laboratory space and reagents, as well as the staff from BL17B beamline of National Center for Protein Sciences Shanghai (NCPSS) at Shanghai Synchrotron Radiation Facility for assistance during data collection. This work was supported by grants from the Netherlands Organization of Scientific Research (NWO) to J.v.d.O. (NWO-TOP, 854.10.003) and to Y. Zhang via a Gravitation grant to the Soehngen Institute for Anaerobic Microbiology (024.002.002), as well as by a fellowship of the European Molecular Biology Organization to D.C.S. (EMBO ALTF 179-2015). C.J. was funded by the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013/ERC grant agreement 309509) and by the Netherlands Organization for Scientific Research (Vidi 864.14.002). Y.W. was funded by grants from the Natural Science Foundation of China (91440201) and the Strategic Priority Research program of the Chinese Academy of Sciences (XDB08010203). Publisher Copyright: {\textcopyright} 2017 Elsevier Inc.",
year = "2017",
month = mar,
day = "16",
doi = "10.1016/j.molcel.2017.01.033",
language = "English",
volume = "65",
pages = "985--998.e6",
journal = "Molecular Cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "6",
}