Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute

Daan C. Swarts, Malwina Szczepaniak, Gang Sheng, Stanley D. Chandradoss, Yifan Zhu, Elizabeth M. Timmers, Yong Zhang, Hongtu Zhao, Jizhong Lou, Yanli Wang*, Chirlmin Joo, John van der Oost

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

22 Citations (Scopus)

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. 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.
Original languageEnglish
Pages (from-to)985-998
JournalMolecular Cell
Volume65
Issue number6
DOIs
Publication statusPublished - 2017

Fingerprint

Argonaute Proteins
Thermus thermophilus
Deoxycytidine
Deoxyguanosine
DNA
Molecular Dynamics Simulation
Fluorescence

Keywords

  • Ago
  • DNA chopping
  • Guide generation
  • Guide loading
  • PAgo
  • Prokaryotic argonaute
  • RNA interference
  • SiDNA
  • Small interfering DNA
  • TtAgo

Cite this

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. / Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute. In: Molecular Cell. 2017 ; Vol. 65, No. 6. pp. 985-998.
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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. 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.",
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Swarts, DC, Szczepaniak, M, Sheng, G, Chandradoss, SD, Zhu, Y, Timmers, EM, Zhang, Y, Zhao, H, Lou, J, Wang, Y, Joo, C & van der Oost, J 2017, 'Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute', Molecular Cell, vol. 65, no. 6, pp. 985-998. https://doi.org/10.1016/j.molcel.2017.01.033

Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute. / 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.

In: Molecular Cell, Vol. 65, No. 6, 2017, p. 985-998.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute

AU - Swarts, Daan C.

AU - Szczepaniak, Malwina

AU - Sheng, Gang

AU - Chandradoss, Stanley D.

AU - Zhu, Yifan

AU - Timmers, Elizabeth M.

AU - Zhang, Yong

AU - Zhao, Hongtu

AU - Lou, Jizhong

AU - Wang, Yanli

AU - Joo, Chirlmin

AU - van der Oost, John

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AB - 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. 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.

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KW - Guide loading

KW - PAgo

KW - Prokaryotic argonaute

KW - RNA interference

KW - SiDNA

KW - Small interfering DNA

KW - TtAgo

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JF - Molecular Cell

SN - 1097-2765

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