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....
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2019-01-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/40836 |
| _version_ | 1828384317165797376 |
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| author | Rani Zananiri Omri Malik Sergei Rudnizky Vera Gaydar Roman Kreiserman Arnon Henn Ariel Kaplan |
| author_facet | Rani Zananiri Omri Malik Sergei Rudnizky Vera Gaydar Roman Kreiserman Arnon Henn Ariel Kaplan |
| author_sort | Rani Zananiri |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-10T05:01:49Z |
| format | Article |
| id | doaj.art-743f74ea77c247089857f091985b97db |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-12-10T05:01:49Z |
| publishDate | 2019-01-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-743f74ea77c247089857f091985b97db2022-12-22T02:01:23ZengeLife Sciences Publications LtdeLife2050-084X2019-01-01810.7554/eLife.40836Synergy between RecBCD subunits is essential for efficient DNA unwindingRani Zananiri0https://orcid.org/0000-0002-0094-4197Omri Malik1Sergei Rudnizky2Vera Gaydar3Roman Kreiserman4Arnon Henn5https://orcid.org/0000-0001-6360-7211Ariel Kaplan6https://orcid.org/0000-0002-9731-6962Faculty of Biology, Technion – Israel Institute of Technology, Haifa, IsraelFaculty of Biology, Technion – Israel Institute of Technology, Haifa, Israel; Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, IsraelFaculty of Biology, Technion – Israel Institute of Technology, Haifa, IsraelFaculty of Biology, Technion – Israel Institute of Technology, Haifa, IsraelFaculty of Biology, Technion – Israel Institute of Technology, Haifa, Israel; Faculty of Physics, Technion – Israel Institute of Technology, Haifa, IsraelFaculty of Biology, Technion – Israel Institute of Technology, Haifa, IsraelFaculty of Biology, Technion – Israel Institute of Technology, Haifa, IsraelThe 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.https://elifesciences.org/articles/40836RecBCDhelicasesingle molecule biophysicsoptical tweezers |
| spellingShingle | Rani Zananiri Omri Malik Sergei Rudnizky Vera Gaydar Roman Kreiserman Arnon Henn Ariel Kaplan Synergy between RecBCD subunits is essential for efficient DNA unwinding eLife RecBCD helicase single molecule biophysics optical tweezers |
| 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 | RecBCD helicase single molecule biophysics optical tweezers |
| url | https://elifesciences.org/articles/40836 |
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