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.
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Daan C
authors.initials: DC
authors.lastname: Swarts
authors.affiliation: Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, 2628 CD Delft, the Netherlands.
authors.forename: Malwina
authors.initials: M
authors.lastname: Szczepaniak
authors.affiliation: Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Sheng
authors.affiliation: Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, 2628 CD Delft, the Netherlands.
authors.forename: Stanley D
authors.initials: SD
authors.lastname: Chandradoss
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WE Wageningen, the Netherlands.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Timmers
authors.affiliation: Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Hongtu
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jizhong
authors.initials: J
authors.lastname: Lou
authors.affiliation: Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: ylwang@ibp.ac.cn.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, 2628 CD Delft, the Netherlands. Electronic address: c.joo@tudelft.nl.
authors.forename: Chirlmin
authors.initials: C
authors.lastname: Joo
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WE Wageningen, the Netherlands. Electronic address: john.vanderoost@wur.nl.
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016373:DNA, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003841:Deoxycytidine / Q000378:metabolism; D003849:Deoxyguanosine / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D000072760:Single Molecule Imaging; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D060565:Argonaute Proteins; D001426:Bacterial Proteins; D016373:DNA, Antisense; D004269:DNA, Bacterial; D003841:Deoxycytidine; D003849:Deoxyguanosine
keywords: DNA chopping; RNA interference; TtAgo; ago; guide generation; guide loading; pAgo; prokaryotic argonaute; siDNA; small interfering DNA