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Synergy between RecBCD subunits is essential for efficient DNA unwinding

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear....

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Autores principales: Zananiri, Rani, Malik, Omri, Rudnizky, Sergei, Gaydar, Vera, Kreiserman, Roman, Henn, Arnon, Kaplan, Ariel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338465/
https://www.ncbi.nlm.nih.gov/pubmed/30601118
http://dx.doi.org/10.7554/eLife.40836
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author Zananiri, Rani
Malik, Omri
Rudnizky, Sergei
Gaydar, Vera
Kreiserman, Roman
Henn, Arnon
Kaplan, Ariel
author_facet Zananiri, Rani
Malik, Omri
Rudnizky, Sergei
Gaydar, Vera
Kreiserman, Roman
Henn, Arnon
Kaplan, Ariel
author_sort Zananiri, Rani
collection PubMed
description The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD’s individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25–40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.
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spelling pubmed-63384652019-01-24 Synergy between RecBCD subunits is essential for efficient DNA unwinding Zananiri, Rani Malik, Omri Rudnizky, Sergei Gaydar, Vera Kreiserman, Roman Henn, Arnon Kaplan, Ariel eLife Biochemistry and Chemical Biology The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD’s individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25–40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state. eLife Sciences Publications, Ltd 2019-01-02 /pmc/articles/PMC6338465/ /pubmed/30601118 http://dx.doi.org/10.7554/eLife.40836 Text en © 2019, Zananiri et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Zananiri, Rani
Malik, Omri
Rudnizky, Sergei
Gaydar, Vera
Kreiserman, Roman
Henn, Arnon
Kaplan, Ariel
Synergy between RecBCD subunits is essential for efficient DNA unwinding
title Synergy between RecBCD subunits is essential for efficient DNA unwinding
title_full Synergy between RecBCD subunits is essential for efficient DNA unwinding
title_fullStr Synergy between RecBCD subunits is essential for efficient DNA unwinding
title_full_unstemmed Synergy between RecBCD subunits is essential for efficient DNA unwinding
title_short Synergy between RecBCD subunits is essential for efficient DNA unwinding
title_sort synergy between recbcd subunits is essential for efficient dna unwinding
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338465/
https://www.ncbi.nlm.nih.gov/pubmed/30601118
http://dx.doi.org/10.7554/eLife.40836
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