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A real-time, transient kinetic study of Drosophila melanogaster Dicer-2 elucidates mechanism of termini-dependent cleavage of dsRNA

Raushan K. Singh, McKenzie Jonely, Evan Leslie, Nick A. Rejali, Rodrigo Noriega, View ORCID ProfileBrenda L. Bass
doi: https://doi.org/10.1101/2020.09.29.319475
Raushan K. Singh
1Department of Biochemistry, University of Utah, Salt Lake City, UT, 84112 USA
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McKenzie Jonely
2Department of Chemistry, University of Utah, Salt Lake City, UT, 84112 USA
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Evan Leslie
1Department of Biochemistry, University of Utah, Salt Lake City, UT, 84112 USA
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Nick A. Rejali
3Department of Pathology, University of Utah, Salt Lake City, UT, 84112 USA
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Rodrigo Noriega
2Department of Chemistry, University of Utah, Salt Lake City, UT, 84112 USA
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Brenda L. Bass
1Department of Biochemistry, University of Utah, Salt Lake City, UT, 84112 USA
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  • ORCID record for Brenda L. Bass
  • For correspondence: bbass@biochem.utah.edu
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SUMMARY

Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3’-overhanging termini. We investigated the transient kinetic mechanism of these reactions using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. We found that ATP binding to dmDcr-2’s helicase domain impacts the kinetics of dsRNA binding and dissociation in a termini-dependent manner, emphasizing the termini-dependent discrimination of dsRNA on a biologically-relevant time-scale. ATP-hydrolysis mediates local unwinding of dsRNA, and directional translocation on unwound single-stranded RNA, which is concurrent with a slow rewinding prior to dsRNA cleavage. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent change in conformational dynamics of the helicase and Platform•PAZ domains in the nanosecond timescale that is correlated with termini-dependent dsRNA cleavage. Our study delineates kinetic events and transient intermediates for a Dicer-catalyzed reaction, thus establishing a framework for understanding other Dicers and how accessory factors modulate the reaction.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted September 30, 2020.
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A real-time, transient kinetic study of Drosophila melanogaster Dicer-2 elucidates mechanism of termini-dependent cleavage of dsRNA
Raushan K. Singh, McKenzie Jonely, Evan Leslie, Nick A. Rejali, Rodrigo Noriega, Brenda L. Bass
bioRxiv 2020.09.29.319475; doi: https://doi.org/10.1101/2020.09.29.319475
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A real-time, transient kinetic study of Drosophila melanogaster Dicer-2 elucidates mechanism of termini-dependent cleavage of dsRNA
Raushan K. Singh, McKenzie Jonely, Evan Leslie, Nick A. Rejali, Rodrigo Noriega, Brenda L. Bass
bioRxiv 2020.09.29.319475; doi: https://doi.org/10.1101/2020.09.29.319475

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